Anti-CLL-1 antibodies and methods of use

ABSTRACT

The present invention relates to anti-CLL-1 antibodies including anti-CLL-1 antibodies comprising a CLL-1 binding domain and a CD3 binding domain (e.g., anti-CLL-1/CD3 T cell dependent bispecific (TDB) antibody) and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of provisional U.S. Application Nos. 62/180,376 filed 16 Jun. 2015, and 62/307,003 filed 11 Mar. 2016, which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 7, 2016, is named P32920-WO_SL.txt and is 62,510 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-CLL-1 antibodies including anti-CLL-1 antibodies comprising a CLL-1 binding domain and a CD3 binding domain (e.g., anti-CLL-1/CD3 T cell dependent bispecific (TDB) antibody) and methods of using the same.

BACKGROUND

Cell proliferative disorders, such as cancer, are characterized by the uncontrolled growth of cell subpopulations. They are the leading cause of death in the developed world and the second leading cause of death in developing countries, with over 12 million new cancer cases diagnosed and 7 million cancer deaths occurring each year. The National Cancer Institute estimates that greater than half a million Americans will die of cancer in 2013, accounting for nearly one out of every four deaths in the country. As the elderly population has grown, the incidence of cancer has concurrently risen, as the probability of developing cancer is more than two-fold higher after the age of seventy. Cancer care thus represents a significant and ever-increasing societal burden.

CLL-1 (also referred to as CLEC12A, MICL, and DCAL2), encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. CLL-1 has been shown to type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain and a short cytoplasmic tail containing an ITIM motif. Further, CLL-1 is present on monocytes and granulocytes in normal peripheral blood and bone marrow (BM), while absent in nonhematological tissues. CLL-1 is also expressed on acute myeloid leukemia (AML), myelodisplastic syndrome (MDS), and chronic myelogenous leukemia (CML) cells. In particular, CLL-1 is a leukemia stem cell (LSC)-associated surface antigen expressed on a fraction of CD34+CD38− AML cells in CD34 positive (CD34+) AML.

Monoclonal antibody (mAb)-based therapy has become an important treatment modality for cancer. Leukemia is well suited to this approach because of the accessibility of malignant cells in the blood, bone marrow, spleen, and lymph nodes and the well-defined immunophenotypes of the various lineages and stages of hematopoietic differentiation that permit identification of antigenic targets. Most studies for acute myeloid leukemia (AML) have focused on CD33. However, responses with the unconjugated anti-CD33 mAb lintuzumab have had modest single agent and activity against AML and failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy.

There is a need in the art for safe and effective agents that target AML including CLL-1 for the diagnosis and treatment of CLL-1-associated conditions, such as cancer. The invention fulfills that need and provides other benefits.

SUMMARY

The invention provides anti-CLL-1 antibodies comprising a CLL-1 binding domain and a CD3 binding domain and methods of using the same. In some embodiments, provided herein are isolated anti-CLL-1 antibodies, wherein the antibody comprises (i) a CLL-1 binding domain, wherein the CLL-1 binding domain binds a CLL-1 epitope and/or binds an overlapping CLL-1 epitope comprising amino acids of SEQ ID NO:49 and does not bind an epitope comprising SEQ ID NO:50 and/or SEQ ID NO:51; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain.

In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope and/or binds an overlapping CLL-1 epitope comprising amino acids of SEQ ID NO:49 and does not bind a CLL-1 epitope comprising SEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope comprising amino acids of SEQ ID NO:49. In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope consisting or consisting essentially of the amino acids of SEQ ID NO:49. In some embodiments, the CLL-1 epitope is determined by hydroxyl radical footprinting.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six hypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7 and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:46 and a light chain variable region comprising the sequence of SEQ ID NO:32

Further, for example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:9; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:33 and a light chain variable region comprising the sequence of SEQ ID NO:32.

In addition, for example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:47; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:48 and a light chain variable region comprising the sequence of SEQ ID NO:32.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32.

Further, for example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:43; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising (a) a heavy chain variable region comprising the sequence of SEQ ID NO:40 and (b) a light chain variable region comprising the sequence of SEQ ID NO:39.

In some embodiments of any of the anti-CLL1 antibodies, the antibody comprises a CLL-1 binding domain has one or more of the following characteristics: a) binds to recombinant human CLL-1; b) binds to recombinant cynomolgus monkey CLL-1; c) binds to endogenous CLL-1 on the surface of human peripheral blood mononucleocytes (PBMCs); d) binds to endogenous CLL-1 on the surface of cynomolgus monkey PBMCs; e) binds to endogenous CLL-1 on the surface of a cancer cell; f) binds to endogenous CLL-1 on the surface of an AML cancer cell; g) binds to endogenous CLL-1 on the surface of HL-60 cells; h) binds to endogenous CLL-1 on the surface of EOL-1 cells; i) binds to CLL-1 comprising a K244Q mutation; j) competes for human CLL-1 binding with R&D clone 687317 antibody; k) binds to endogenous human CLL-1 with a Kd of less than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM; l) binds to recombinant human CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM; and/or m) binds to recombinant cynomolgus monkey CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM, less than 2 nM, or less than 1 nM.

In some embodiments of any of the anti-CLL1 antibodies, the antibody comprises a CD3 binding domain which binds a CD3 epitope comprising and/or consisting of Glu6 of human CD3ε polypeptide. In certain aspects, the CD3 epitope further comprises one or more additional amino acid residues selected from the group consisting of Gln1, Asp2, and Met7 of CD3. In some embodiments of any of the anti-CLL1 antibodies, the antibody comprises a CD3 binding domain which binds a CD3 epitope comprising and/or consisting of Gln1, Asp2, and Glu6 of human CD3ε polypeptide. In some embodiments of any of the anti-CLL1 antibodies, the antibody comprises a CD3 binding domain which binds a CD3 epitope comprising and/or consisting of Gln1, Asp2, Glu6, and Met7 of human CD3ε polypeptide. In another aspect, the CD3 epitope does not comprise amino acid residue Glu5 of human CD3ε polypeptide. In a further aspect, the CD3 epitope does not comprise amino acid residues Gly3 and Glu5 of human CD3ε polypeptide. In certain aspects, the CD3 epitope consists of amino acid residues Gln1, Asp2, Glu6, and Met7 of human CD3ε polypeptide.

In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 50 nM and greater than 1 nM. In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 1 nM and greater than 0.1 nM. In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 0.1 nM and greater than 0.01 nM. In some embodiments, the affinity of the CD3 binding arm is determined by Biacore. In some embodiments, the human CD3 is hCD3εγ. In some embodiments, the human CD3 is hCD3ε 1-27 Fc.

In some embodiments of any of the anti-CLL1 antibodies, the antibody comprises a CD3 binding domain comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:91; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:91; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32.

In some embodiments of any of the anti-CLL1 antibodies, the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:82 and a light chain variable region comprising the sequence of SEQ ID NO:83. For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:82 and a light chain variable region comprising the sequence of SEQ ID NO:83.

In some embodiments of any of the anti-CLL1 antibodies, the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32.

In some embodiments of any of the anti-CLL1 antibodies, the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, the anti-CLL-1 antibody comprises a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:85. For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:85.

In some embodiments of any of the anti-CLL1 antibodies, the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:93. For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:93.

In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody is a monoclonal, human, humanized, or chimeric antibody. In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody is an IgG antibody. In some embodiments of any of the anti-CLL-1 antibodies, the antibody is a bispecific antibody. In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody is an antibody fragment that binds CLL-1 and CD3. In some embodiments, the anti-CLL-1 antibody fragment is a Fab, Fab′-SH, Fv, scFv, and/or (Fab′)₂ fragment. In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody is a full-length antibody.

In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody comprises an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation is a substitution mutation.

In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody comprises reduced effector function. In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody comprises a substitution mutation is at amino acid residue N297, L234, L235, D265, and/or P329G according to EU numbering. In some embodiments, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G according to EU numbering. In some embodiments, the anti-CLL-1 antibody comprises an N297G substitution mutation at amino acid residue 297 according to EU numbering. In some embodiments, the anti-CLL-1 antibody comprises an L234A, L235A, and P329G mutation at amino acid residues 234, 235, and 329 according to EU numbering.

In some embodiments of any of the multispecific anti-CLL-1 antibodies, the anti-CLL-1 antibody comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH1₁) domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, a second CH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3 (CH3₂) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some embodiments, the CH3₁ and CH3₂ domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH3₁ domain is positionable in the cavity or protuberance, respectively, in the CH3₂ domain. In some embodiments, the CH3₁ and CH3₂ domains meet at an interface between said protuberance and cavity. In some embodiments, the CH2₁ and CH2₂ domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH2₁ domain is positionable in the cavity or protuberance, respectively, in the CH2₂ domain. In some embodiments, the CH2₁ and CH2₂ domains meet at an interface between said protuberance and cavity.

In some embodiments of any of the anti-CLL-1 antibodies, (a) the CD3 binding domain comprises a Fc domain, wherein the Fc domain comprises T366S, L368A, Y407V, and N297G substitution mutations according EU numbering and (b) the CLL-1 binding domain comprises a Fc domain, wherein the Fc domain comprises T366W and N297G substitution mutations according EU numbering. In some embodiments of any of the anti-CLL-1 antibodies, (a) the CD3 binding domain comprises a Fc domain, wherein the Fc domain comprises L234A, L235A, P329G, T366S, L368A, and Y407V, substitution mutations according EU numbering and (b) the CLL-1 binding domain comprises a Fc domain, wherein the Fc domain comprises L234A, L235A, P329G, and T366W substitution mutations according EU numbering.

Further provided herein are isolated nucleic acids encoding an anti-CLL-1 antibody described herein. Also provided herein are vectors comprising an isolated nucleic acid encoding an anti-CLL-1 antibody described herein. Provided herein are also host cells comprising a vector comprising an isolated nucleic acid encoding an anti-CLL-1 antibody described herein. Also provided are methods of producing an anti-CLL-1 antibody described herein, the method comprising culturing a host cell comprising a vector comprising an isolated nucleic acid encoding an anti-CLL-1 antibody described herein in a culture medium.

In addition, provided herein are pharmaceutical compositions comprising an anti-CLL-1 antibody described herein.

In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody has a cell killing EC₅₀ of less than about 200 ng/mL, e.g., less than about any of 150, 100, 50, 25, 20, or 15 ng/mL. In some embodiments, the cell killing is human autologous CD14+. In some embodiments, the cell killing is cell line cell killing, e.g., U937 cell line, HL-60 cell line, PL-21 cell line, NOMO-1 cell line, EOL-1 cell line, THP-1 cell line, ML-2 cell line, Molm-13 cell line. In some embodiments of any of the multispecific anti-CLL-1 antibodies, the anti-CLL-1 antibody has a cytotoxic T cell activation EC₅₀ is less than about any of 50 ng/mL, e.g., less than about any of 25 ng/mL or 20 ng/mL. In some embodiments, cytotoxic T cell activation is measured by % of CD69+CD25+ T cells in CD8+ T cells.

Provided herein are anti-CLL-1 antibodies as described herein for use as a medicament. Provided herein are anti-CLL-1 antibody described herein for use in treating or delaying progression of a cell proliferative disorder or an autoimmune disorder in a subject in need thereof. Provided herein are anti-CLL-1 antibodies as described herein for use in enhancing immune function in a subject having a cell proliferative disorder or an autoimmune disorder. In some embodiments, the cell proliferative disorder is a cancer. In some embodiments, the cell proliferative disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and/or myelodysplastic syndrome (MDS). In some embodiments, the cell proliferative disorder is CLL-1 positive. In some embodiments of any of the cell proliferative disorders, the cell proliferative disorder is resistant to treatment with an anti-AML antibody drug conjugate (e.g., anti-CLL1, anti-CD123, and/or anti-CD33 antibody drug conjugate). In some embodiments, the drug of the antibody drug conjugate is a DNA damaging agent (e.g., PBD).

Provided herein are uses of any of the anti-CLL-1 antibody described herein in the manufacture of a medicament for treating or delaying progression of a cell proliferative disorder or an autoimmune disorder. Provided herein are uses of any of the anti-CLL-1 antibody described herein in the manufacture of a medicament for enhancing immune function in a subject having a cell proliferative disorder or an autoimmune disorder. In some embodiments, the cell proliferative disorder is a cancer. In some embodiments, the cell proliferative disorder is AML, CML, and/or MDS. In some embodiments, the cell proliferative disorder is CLL-1 positive. In some embodiments of any of the cell proliferative disorders, the cell proliferative disorder is resistant to treatment with an anti-AML antibody drug conjugate (e.g., anti-CLL1, anti-CD123, and/or anti-CD33 antibody drug conjugate). In some embodiments, the drug of the antibody drug conjugate is a DNA damaging agent (e.g., PBD).

Provided herein are methods of treating or delaying the progression of a cell proliferative disorder or an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject an anti-CLL-1 antibody described herein. Provided herein are methods of enhancing immune function in a subject having a cell proliferative disorder or an autoimmune disorder, the method comprising administering to the subject an effective amount of an anti-CLL-1 antibody described herein. In some embodiments, the cell proliferative disorder is a cancer. In some embodiments, the cell proliferative disorder is AML, CML, and/or MDS. In some embodiments, the cell proliferative disorder is CLL-1 positive. In some embodiments of any of the cell proliferative disorders, the cell proliferative disorder is resistant to treatment with an anti-AML antibody drug conjugate (e.g., anti-CLL1, anti-CD123, and/or anti-CD33 antibody drug conjugate). In some embodiments, the drug of the antibody drug conjugate is a DNA damaging agent (e.g., PBD).

In some embodiments of any of the methods, the anti-CLL-1 antibody binds to (a) a CD3 molecule located on an immune effector cell and (b) a CLL-1 molecule located on a cell. In some embodiments, the anti-CLL-1 antibody activates the immune effector cell following binding to (a) and (b). In some embodiments of any of the methods, the activated immune effector cell is capable of exerting a cytotoxic effect and/or an apoptotic effect on the target cell.

In some embodiments of any of the methods, the method further comprises administering to the subject a PD-1 axis binding antagonist or an additional therapeutic agent. In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

In some embodiments of any of the methods, the method further comprises administering to the subject a glucocorticoid. In some embodiments, the glucocorticoid is dexamethasone.

In some embodiments of any of the methods, the method is used in combination with stem cell transplant.

In some embodiments of any of the methods, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent comprises cytarabine and/or daunorubicin. In some embodiments, the second therapeutic agent comprises cytarabine. In some embodiments, the second therapeutic agent comprises an anti-AML antibody drug conjugate (e.g., anti-CLL1, anti-CD123, and/or anti-CD33 antibody drug conjugate).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B shows alignment of the light chain variable region sequences (SEQ ID NOS 30, 37, 35 and 41, respectively, in order of appearance) (A) and heavy chain variable region sequences (SEQ ID NOS 31, 38, 36 and 42, respectively, in order of appearance) (B) of murine (m) 6E7, m21C9, m20B1, and m28H12.

FIG. 2A-B shows alignment of the light chain variable region sequences (SEQ ID NOS 109, 30, 32 and 32, respectively, in order of appearance) (A) and heavy chain variable region sequences (SEQ ID NOS 110, 31, 33 and 34, respectively, in order of appearance) (B) of K1H1, m6E7, humanized (h) 6E7.L4H1e, and h6E7.L4H1e.A54.

FIG. 3A-B shows alignment of the light chain variable region sequences (SEQ ID NOS 109, 37 and 39, respectively, in order of appearance) (A) and heavy chain variable region sequences (SEQ ID NOS 110, 38 and 40, respectively, in order of appearance) (B) of K1H1, m21C9, and h21C9.L2H3.

FIG. 4A-C shows in vitro killing characterization of anti-CLL-1/CD3 TDBs. (A) shows percent killing of EOL-1 at various concentrations of TDB, μg/mL, (gD, h6E7 (6E7.L4H1e), h6E7D (6E7.L4H1eD54), h6E7A (6E7.L4H1eA54), m20B1, and m28H12 in combination with 38E4v1). (B) and (C) show percent cell killing (PBMC, EOL-1, and THP-1) and CD8 (CD8⁺, CD69⁺, CD25⁺) T cell activation, respectively, using gD, 6E7 (6E7.L4H1e), and h21C9 (h21C9.L2H3) in combination with 38E4v1 at various concentrations of TDBs (μg/mL).

FIG. 5A-D shows potency of anti-CLL-1 TDBs on various AML tumor cell lines comparing percent survival normalized to drug at various TDB concentrations (μg/mL) using gD×38E4v1, h21C9 (h21C9.L2H3)×38E4v1, and h6E7 (6E7.L4H1e)×38E4v1.

FIG. 6A-B shows in vitro characterization of h6E7 (6E7.L4H1e)×38E4v1 and h21C9 (h21C9.L2H3)×38E4v1 is killing of cynomolgus CD14+ cells in the presence of autologous cynomolgus PBMCs containing all subtypes of T cells and CD8 (CD8⁺, CD69⁺, CD25⁺) T cell activation at various TDB concentrations (μg/mL).

FIG. 7A-B shows in vitro potency of anti-CLL-1 TDB on AML patient bone marrow using h6E7 (6E7.L4H1e)×38E4v1 and gD×38E4v1.

FIG. 8A-D shows in vitro characterization of anti-gD×38E4v1 control, anti-CLL-1 TDBs with low affinity CD3 arm (h6E7 (6E7.L4H1e)×40G5c), high affinity anti-CD3 arm (h6E7 (6E7.L4H1e)×38E4v1), and very high affinity anti-CD3 arm (h6E7 (6E7.L4H1e)×38E4v11) using HL60, THP-1, and EOL-1 cell lines). FIG. 8E-G shows in vitro characterization of variants of h6E7.L4H1e N54-A or -S or -E or -Q or -D humanized Fab paired with low affinity anti-CD3 Fab (h40G5c) or high affinity anti-CD3 Fab (38E4v1). FIG. 8H-I shows CD8 T cell activation in response to h6E7N54 variants paired with the high affinity anti-CD3 arm (38E4v1) or the low affinity anti-CD3 arm (h40G5c).

FIG. 9A-B shows expression of hCLL-1 and hCD3e transgenes in double BAC-Tg (hCL-1/hCD3ε) mice.

FIG. 10A-D shows expression of hCLL-1 and hCD3e transgenes in heterozygous double BAC-Tg (hCL-1/hCD3ε) mice compared to homozygous parents.

FIG. 11 shows ex vivo activation of CD8⁺ T cells in double BAC-Tg (hCL-1/hCD3e) treated with anti-CLL-1 TDBs, gD×38E4v1, 6E7 (6E7.L4H1e)×38E4v1, N54A (6E7.L4H1eA54)×38E4v1, and N54A (6E7.L4H1eA54)×40G5 (40G5c).

FIG. 12 shows in vivo activation of CD8⁺ T cells double BAC-Tg (hCL-1/hCD3e) treated with anti-CLL-1 TDBs, gD×38E4v1, gD×40G5c, h6E7A (6E7.L4H1eA54)×40G5c, h6E7A (6E7.L4H1eA54)×38E4v1, and h6E7A (6E7.L4H1eA54)×38E4v11.

FIG. 13A-B shows in vivo depletion of hCLL-1⁺ cells in hCLL-1/hCD3e BAC-Tg mice treated with anti-CLL-1 TDBs with anti-CLL-1 TDBs, gD×CD3 High (38E4v1), gD×CD3 Low (40G5c), CLL1(A) (6E7.L4H1eA54)×CD3 Low (40G5c), CLL1(A) (6E7.L4H1eA54)×CD3 High (38E4v1), and CLL1(A) (6E7.L4H1eA54)×CD3 Very High (38E4v11).

FIG. 14A-B shows expression profile of CLL-1 (A) and (B) and CD33 (B) on normal and AML human leukocytes.

FIG. 15A-C shows impact on hematopoiesis following treatment of human bone marrow cells with anti-CLL-1 h6E7A (6E7.L4H1eA5)×38E4v1 TDB.

FIG. 16A-B shows alignment of the light chain variable region sequences (SEQ ID NOS 85, 93 and 83, respectively, in order of appearance) (A) and heavy chain variable region sequences (SEQ ID NOS 84, 84 and 82, respectively, in order of appearance) (B) of 38E4v1, 38E4v11, and 40G5C.

FIG. 17A-D shows pharmacokinetic (PK) data in SCID.beige mice (A and B) and hCLL-1/hCD3e BAC-Tg mice (C and D) treated with anti-CLL-1 TDBs.

FIG. 18A-C shows pharmacodynamics (PD) data in peripheral blood (A), bone marrow (B), and spleen (C) of hCLL-1/hCD3e BAC-Tg mice treated with h6E7A (6E7.L4H1eA54)×h40G5c TDB.

FIG. 19A-C shows PD data in peripheral blood (A), bone marrow (B), and spleen (C) of hCLL-1/hCD3e BAC-Tg mice treated with h6E7N54A (6E7.L4H1eA54)×38E4v1 TDB.

FIG. 20A-C shows PD data in peripheral blood (A), bone marrow (B), and spleen (C) of hCLL-1/hCD3e BAC-Tg mice treated with h6E7N54A (6E7.L4H1eA54)×38E4v11 TDB.

FIG. 21A-F shows upregulation of CD69 on mouse CD8+ cells correlated with the time point associated with maximal target diminution in hCLL-1/hCD3e BAC-Tg mice treated with h6E7A (6E7.L4H1eA54)×h40G5c TDB (A and D), h6E7N54A (6E7.L4H1eA54)×38E4v1 TDB (B and E), and h6E7N54A (6E7.L4H1eA54)×38E4v11 TDB (C and F).

FIG. 22A-B shows myeloid cell reduction in hCLL-1/hCD3e BAC-Tg mice treated with h6E7N54A (6E7.L4H1eA54)×h40G5c and anti-PD-L1. FIG. 22C-D shows upregulation of PD-L1 on monocytes and granulocytes in hCLL-1/hCD3e BAC-Tg mice treated with anti-CLL-1/CD3 TDB antibody.

FIG. 23A-D shows myeloid cell numbers or CLL-1-positive cell numbers in cynomolgus monkeys treated with the low affinity anti-CLL-1/CD3 TDB antibody ((6E7.L4H1eA54)×h40G5c).

FIG. 24 shows circulating lymphocyte numbers in cynomolgus monkeys treated with the low affinity anti-CLL-1/CD3 TDB antibody ((6E7.L4H1eA54)×h40G5c).

FIG. 25 shows PK parameters in cynomolgus monkeys treated with anti-CLL-1/CD3 TDB antibodies.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “CLL-1,” as used herein, refers to any native, mature CLL-1 which results from processing of a CLL-1 precursor protein in a cell. The term includes CLL-1 from any vertebrate source, including mammals such as primates (e.g. humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of CLL-1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CLL-1 protein sequence is shown in SEQ ID NO:1. In some embodiments, the human CLL-1 protein sequence comprises the K244Q SNP (SEQ ID NO:1, wherein K244 is Q). The amino acid sequence of an exemplary extracellular domain is the amino acids of SEQ ID NO:2. The amino acid sequence of an exemplary C-type lectin like domain (CTLD) is the amino acids of SEQ ID NO:3. The amino acid sequence of an exemplary cynomolgus monkey CLL-1 protein is shown in SEQ ID NO:4.

The term “glycosylated forms of CLL-1” refers to naturally occurring forms of CLL-1 that are post-translationally modified by the addition of carbohydrate residues.

The term “cluster of differentiation 3” or “CD3,” as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses “full-length,” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length, and human CD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length.

The terms “anti-CLL-1 antibody” and “an antibody that binds to CLL-1” refer to an antibody that is capable of binding CLL-1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CLL-1. In one embodiment, the extent of binding of an anti-CLL-1 antibody to an unrelated, non-CLL-1 protein is less than about 10% of the binding of the antibody to CLL-1 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CLL-1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ε 10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-CLL-1 antibody binds to an epitope of CLL-1 that is conserved among CLL-1 from different species.

The terms “anti-CD3 antibody” and “an antibody that binds to CD3” refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD3. In one embodiment, the extent of binding of an anti-CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD3 has a dissociation constant (Kd) of 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

By “binding domain” is meant a part of a compound or a molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include but are not limited to antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab′2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules having an identified binding partner.

The term “epitope” refers to the particular site on an antigen molecule to which an antibody binds. In some embodiments, the particular site on an antigen molecule to which an antibody binds is determined by hydroxyl radical footprinting (e.g., CLL-1 binding domain). In some embodiments, the particular site on an antigen molecule to which an antibody binds is determined by crystallography (e.g., CD3 binding domain).

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors; and B cell activation.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CLL-1 antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

“Isolated nucleic acid encoding an anti-CD3 antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

As used herein, “delaying progression” of a disorder or disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease or disorder (e.g., a cell proliferative disorder, e.g., cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

By “reduce” or “inhibit” is meant the ability to cause an overall decrease, for example, of 20% or greater, of 50% or greater, or of 75%, 85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit can refer to the effector function of an antibody that is mediated by the antibody Fc region, such effector functions specifically including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1| and calicheamicin omega|1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETAN, alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

The term “immunosuppressive agent” as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL® methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor (TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; transforming growth factor-beta (TGF-beta); streptodornase; RNA or DNA from the host; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies and zTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol., 23:113-5 (2002) and see also definition below); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al., Science, 261: 1328-30 (1993); Mohan et al., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Some preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

The term “PD-1 axis binding antagonist” refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis—with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist is MDX-1106 (nivolumab) described herein. In another specific aspect, a PD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. In another specific aspect, a PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another specific aspect, a PD-1 binding antagonist is AMP-224 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1, B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, an anti-PD-L1 antibody is YW243.55.S70 described herein. In another specific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD-L1 antibody is MPDL3280A (CAS Registry Number 1380723-44-3) described herein. In still another specific aspect, an anti-PD-L1 antibody is MED14736 described herein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.

The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. In some embodiments, the cancer is a CLL-1 positive cancer. Examples of cancer including CLL-1 positive cancers include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia (APL), chronic myeloproliferative disorder, thrombocytic leukemia, precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursor T-cell acute lymphoblastic leukemia (preT-ALL), multiple myeloma (MM), mast cell disease, mast cell leukemia, mast cell sarcoma, myeloid sarcomas, lymphoid leukemia, and undifferentiated leukemia. In some embodiments, the cancer is myeloid leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML).

The term “CLL-1-positive cell” refers to a cell that expresses CLL-1 on its surface.

The term “CLL-1-positive cancer” refers to a cancer comprising cells that express CLL-1 on their surface. In some embodiments, expression of CLL-1 on the cell surface is determined, for example, using antibodies to CLL-1 in a method such as immunohistochemistry, FACS, etc. Alternatively, CLL-1 mRNA expression is considered to correlate to CLL-1 expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

It is understood that aspects and variations of the invention described herein include “consisting of” and/or “consisting essentially of” aspects and variations.

II. Compositions and Methods

In one aspect, the invention is based, in part, on anti-CLL-1 antibodies. In certain embodiments, the anti-CLL-1 antibodies comprising a CLL-1 binding domain and a CD3 binding domain are provided. In certain embodiments, the anti-CLL-1 antibodies are anti-CLL-1 T cell dependent bispecific (TDB) antibodies. Antibodies of the invention are useful, e.g., for the diagnosis or treatment of cancer such as acute myeloid leukemia (AML).

A. Exemplary Anti-CLL-1 Antibodies

The invention provides anti-CLL-1 antibodies comprising a CLL-1 binding domain and a CD3 binding domain and methods of using the same. In some embodiments, provided herein are isolated anti-CLL-1 antibodies, wherein the antibody comprises (i) a CLL-1 binding domain, wherein the CLL-1 binding domain binds a CLL-1 epitope and/or binds an overlapping CLL-1 epitope comprising amino acids of SEQ ID NO:49 and does not bind an epitope comprising SEQ ID NO:50 and/or SEQ ID NO:51; and (ii) a CD3 binding domain, wherein the CD3 binding domain binds a human CD3ε polypeptide and a cyno CD3ε polypeptide, the CD3 binding domain binds to a CD3 epitope within a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87) of human CD3ε, and amino acid residue Glu5 of the human CD3ε polypeptide is not required for binding of the CD3 binding domain.

a) CLL-1 Binding Domains of Anti-CLL1 Antibodies

In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope and/or binds a CLL-1 overlapping epitope comprising amino acids of SEQ ID NO:49 and does not bind a CLL-1 epitope comprising SEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope comprising amino acids of SEQ ID NO:49. In some embodiments, the CLL-1 binding domain of the anti-CLL-1 antibody binds a CLL-1 epitope consisting or consisting essentially of the amino acids of SEQ ID NO:49. In some embodiments, the CLL-1 epitope is determined by hydroxyl radical footprinting. In some embodiments, the CLL-1 epitope as determined by hydroxyl radical footprinting has a ratio of [rate constant of the antigen]/[rate constant of the antigen and antibody complex] greater than about any of 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, the a CLL-1 epitope as determined by hydroxyl radical footprinting has a ratio of [rate constant of the antigen]/[rate constant of the antigen and antibody complex] greater than about 2.0.

Hydroxyl radical footprinting may be performed as described in the Examples. For example, samples are exposed to hydroxyl radicals for intervals of 0, 10, 15, and 20 milliseconds (ms) using the X28c Beam line at the Brookhaven National Laboratory. The labeled samples may be subjected to deglycosylation using PNGase F. The samples may be precipitated using Trichloroacetic acid in acetone, and subjected to LC-MS analysis. The samples may be then subjected to reduction and alkylation, digestion using Trypsin, followed by liquid chromatography coupled with high-resolution mass spectrometry (LC-MS). The MS data may be analyzed using ProtMapMS, resulting in dose response plots for each peptide. Results from the free antigen may be compared against each of the complex forms. A homology-based model of the antigen may be generated using Swiss-Model software, and the solvent protected regions may be mapped for each of the three complexes. The selected ion chromatograms (SIC) may be extracted and integrated for the unoxidized and all oxidized forms of peptide ion (with particular m/z). These peak area values may be used to characterize reaction kinetics in the form of dose response (DR) plots, which measure the loss of intact peptide as a function of the hydroxyl radical exposure. The solvent protected regions in the complex experience gradual oxidation reaction as opposed to the free antigen, and the differences in the rate of oxidation (called rate constant, RC) may serve to highlight the location of the epitope (e.g., CLL-1 epitope).

In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to recombinant human CLL-1. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to recombinant cynomolgus monkey CLL-1. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of human peripheral blood mononucleocytes (PBMCs). In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of cynomolgus monkey PBMCs. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of a cancer cell. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of an AML cancer cell. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of HL-60 cells. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous CLL-1 on the surface of EOL-1 cells. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to CLL-1 comprising a K244Q mutation (SEQ ID NO:1 with K244Q). In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds a CLL-1 epitope and/or binds an overlapping CLL-1 epitope comprising amino acids of SEQ ID NO:49. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain does not bind a CLL-1 epitope comprising SEQ ID NO:50 and/or SEQ ID NO:51. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain competes for human CLL-1 binding with R&D System Clone 687317 antibody. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to endogenous human CLL-1 with a Kd of less than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to recombinant human CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM. In some embodiments of any of the anti-CLL1 antibodies, the CLL-1 binding domain binds to recombinant cynomolgus monkey CLL-1 with a Kd of less than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the Kd is determined by any method described herein, in particular the examples. In some embodiments, Kd is determined by BIACORE. In some embodiments, Kd is determined by CLL-1 immobilized at a low density.

In some embodiments of any of the anti-CLL-1 antibodies, the anti-CLL-1 antibody has a cell killing EC₅₀ of less than about 200 ng/mL, e.g., less than about any of 150, 100, 50, 25, 20, or 15 ng/mL. In some embodiments, the cell killing is human autologous CD14+. In some embodiments, the cell killing is cell line cell killing, e.g., U937 cell line, HL-60 cell line, PL-21 cell line, NOMO-1 cell line, EOL-1 cell line, THP-1 cell line, ML-2 cell line, Molm-13 cell line. In some embodiments of any of the multispecific anti-CLL-1 antibodies, the anti-CLL-1 antibody has a cytotoxic T cell activation EC₅₀ is less than about any of 50 ng/mL, e.g., less than about any of 25 ng/mL or 20 ng/mL. In some embodiments, cytotoxic T cell activation is measured by % of CD69+CD25+ T cells in CD8+ T cells.

In some embodiments, the characteristics of the CLL-1 binding domain and/or CLL-1 antibody are determined as described herein in the Examples below.

CLL-1 Binding Domain 6E7 and Other Embodiments

In some embodiments of any of the CLL-1 antibodies, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:9. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:47. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:11. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:43. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:44.

In one aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10. In one embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:10. In another embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:10 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In a further embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:10, HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:45. In a further embodiment, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:9. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:47. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:11. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:43. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:44.

In another aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In one embodiment, the CLL-1 binding domain comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:10; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:9; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:47; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:43; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In any of the above embodiments, an anti-CLL-1 antibody comprising a CLL-1 binding domain is humanized. In one embodiment, an anti-CLL-1 antibody comprising a CLL-1 binding domain comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is the human VL kappa I consensus (VL_(KI)) framework and/or the VH framework VH₁. In certain embodiments, the human acceptor framework is the human VL kappa I consensus (VL_(KI)) framework and/or the VH framework VH₁ comprising any one of the following mutations.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but a CLL-1 binding domain comprising that sequence retains the ability to bind to CLL-1. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CLL-1 antibody comprising the CLL-1 binding domain comprises the VH sequence of SEQ ID NO:31, SEQ ID NO:33, and/or SEQ ID NO:34, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:9. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:47. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:11. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:43. In some embodiments, HVR-H2 comprises the amino acid sequences of SEQ ID NO:44.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain is provided, wherein the CLL-1 binding domain comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:30 and/or SEQ ID NO:32. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:30 and/or SEQ ID NO:32 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but a CLL-1 binding domain comprising that sequence retains the ability to bind to CLL-1. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:30 and/or SEQ ID NO:32. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:30 and/or SEQ ID NO:32. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CLL-1 antibody comprising the CLL-1 binding domain comprises the VL sequence of SEQ ID NO:30 and/or SEQ ID NO:32, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain is provided, wherein the CLL-1 binding domain comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, the CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:31 and SEQ ID NO:30, respectively, including post-translational modifications of those sequences. In one embodiment, the CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:32, respectively, including post-translational modifications of those sequences. In one embodiment, the CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:34 and SEQ ID NO:32, respectively, including post-translational modifications of those sequences. In one embodiment, the CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:46 and SEQ ID NO:32, respectively, including post-translational modifications of those sequences. In one embodiment, the CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:48 and SEQ ID NO:32, respectively, including post-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to the same epitope as CLL-1 binding domain provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as a CLL-1 binding domain comprising a VH sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:46 and/or SEQ ID NO:48 and a VL sequence of SEQ ID NO:30 and/or SEQ ID NO:32, respectively.

Provided herein are an anti-CLL-1 antibodies comprising a CLL-1 binding domain comprising a light chain variable domain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabat numbering as depicted in FIG. 2A and a heavy chain variable domain comprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabat numbering as depicted in FIG. 2B. In some embodiments, the anti-CLL-1 antibody comprising the CLL-1 binding domain comprises a light chain variable domain comprising the HVR1-LC, HVR2-LC and/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence as depicted in FIG. 2A. In some embodiments, the antibody comprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC sequence as depicted in FIG. 2B.

In a further aspect of the invention, an anti-CLL-1 antibody comprising a CLL-1 binding domain according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-CLL-1 antibody comprising a CLL-1 binding domain is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. In another embodiment, the anti-CLL-1 antibody comprising a CLL-1 binding domain is a substantially full length antibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody class or isotype as defined herein.

CLL-1 Binding Domain 21C9 and Other Embodiments

In some embodiments, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In one aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23. In one embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:23. In another embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:23 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:20. In a further embodiment, the CLL-1 binding domain comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:23, HVR-L3 comprising the amino acid sequence of SEQ ID NO:20, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:22. In a further embodiment, the CLL-1 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23.

In another aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20. In one embodiment, the CLL-1 binding domain comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:23; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In another aspect, the invention provides an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In any of the above embodiments, an anti-CLL-1 antibody comprising a CLL-1 binding domain is humanized. In one embodiment, an anti-CLL-1 antibody comprising a CLL-1 binding domain comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is the human VL kappa I consensus (VL_(KI)) framework and/or the VH framework VH₁. In certain embodiments, the human acceptor framework is the human VL kappa I consensus (VL_(KI)) framework and/or the VH framework VH₁ comprising any one of the following mutations.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:38 and/or SEQ ID NO:40. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:38 and/or SEQ ID NO:40 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but a CLL-1 binding domain comprising that sequence retains the ability to bind to CLL-1. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:38 and/or SEQ ID NO:40. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:38 and/or SEQ ID NO:40. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CLL-1 antibody comprising a CLL-1 binding domain comprises the VH sequence of SEQ ID NO:38 and/or SEQ ID NO:40, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:37 and/or SEQ ID NO:39. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:37 and/or SEQ ID NO:39 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but a CLL-1 binding domain comprising that sequence retains the ability to bind to CLL-1. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:37 and/or SEQ ID NO:39. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:37 and/or SEQ ID NO:39. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CLL-1 antibody comprising a CLL-1 binding domain comprises the VL sequence of SEQ ID NO:37 and/or SEQ ID NO:39, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.

In another aspect, an anti-CLL-1 antibody comprising a CLL-1 binding domain is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, the anti-CLL-1 antibody comprising a CLL-1 binding domain comprises the VH and VL sequences in SEQ ID NO:38 and SEQ ID NO:37, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:40 and SEQ ID NO:39, respectively, including post-translational modifications of those sequences.

In a further aspect, provided are herein are anti-CLL-1 antibody comprising a CLL-1 binding domain that bind to the same epitope as an anti-CLL-1 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising a VH sequence of SEQ ID NO:38 and/or SEQ ID NO:40 and a VL sequence of SEQ ID NO:37 and/or SEQ ID NO:39, respectively.

Provided herein are an anti-CLL-1 antibody comprising a CLL-1 binding domain comprising a light chain variable domain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabat numbering as depicted in FIG. 3A and a heavy chain variable domain comprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabat numbering as depicted in FIG. 3B. In some embodiments, the anti-CLL-1 antibody comprising the CLL-1 binding domain comprises a light chain variable domain comprising the HVR1-LC, HVR2-LC and/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence as depicted in FIG. 3A. In some embodiments, the anti-CLL-1 antibody comprising the CLL-1 binding domain comprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC sequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CLL-1 antibody comprising a CLL-1 binding domain according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-CLL-1 antibody comprising a CLL-1 binding domain is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody class or isotype as defined herein.

b) CD3 Binding Domains of Anti-CLL1 Antibodies

In some embodiments, the CD3 binding domain of the anti-CLL-1 antibody binds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide. In some embodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3ε polypeptide or a cyno CD3ε polypeptide, respectively. In some embodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3γ polypeptide or a cyno CD3γ polypeptide, respectively. In certain embodiments, an anti-CLL-1 antibody is provided comprising a CD3 binding domain which binds to an epitope within a fragment of CD3 (e.g., human CD3ε) consisting of amino acids 1-26 or 1-27 of human CD3ε. In some embodiments, the anti-CLL-1 antibody is a bispecific antibody. In some embodiments, the anti-CLL-1 antibody is a bispecific IgG antibody.

In some embodiments, CD3 binding domain binds the human CD3ε polypeptide with a Kd of 250 nM or lower. In some embodiments, the CD3 binding domain binds the human CD3ε polypeptide with a Kd of 100 nM or lower. In some embodiments, the CD3 binding domain binds the human CD3ε polypeptide with a Kd of 15 nM or lower. In some embodiments, CD3 binding domain binds the human CD3ε polypeptide with a Kd of 10 nM or lower. In some embodiments, CD3 binding domain binds the human CD3ε polypeptide with a Kd of 5 nM or lower. In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 50 nM and greater than 1 nM. In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 1 nM and greater than 0.1 nM. In some embodiments, the CD3 binding arm of the anti-CLL1 antibody binds human CD3 with an affinity less than 0.1 nM and greater than 0.01 nM. In some embodiments, the affinity of the CD3 binding arm is determined by Biacore. In some embodiments, the human CD3 is hCD3εγ. In some embodiments, the human CD3 is hCD3ε 1-27 Fc.

In some embodiments, the CD3 binding domain of the anti-CLL-1 antibody binds contacts with amino acids of human CD3ε at a distance of 3.5 Angstroms, 3.25 Angstroms, 3.00 Angstroms, 2.75 Angstroms, or less. In certain embodiments, the CD3 binding domain is provided that binds to an epitope consisting of one, two, three, four, or five amino acids of human CD3ε at a distance of 3.5 Angstroms, 3.25 Angstroms, 3.00 Angstroms, 2.75 Angstroms or less. In one embodiment, the CD3 binding domain of the anti-CLL-1 antibody makes unique contacts with amino acids of human CD3ε at a distance of 3.5 Angstroms or less. In certain embodiments, the CD3 binding domain is provided that binds to an epitope consisting of one, two, three, four, or five amino acids of human CD3ε at a distance of 3.5 Angstroms or less. For example, in certain embodiments, the CD3 binding domain is provided that binds to an epitope consisting of amino acids of human CD3ε selected from Gln1, Asp2, Asn4, Glu6, and Met7. In one particular embodiment, the CD3 binding domain binds to an epitope that specifically includes Glu6. In certain other embodiments, the CD3 binding domain is provided that does not bind to an epitope that includes human CD3ε amino acid Glu5. In certain other embodiments, the CD3 binding domain is provided that does not bind to an epitope that includes human CD3ε amino acids Gly3 and Glu5.

A CD3 epitope may be determined by the CD3 binding domain binding to peptide fragments of the epitope. Alternatively, a CD3 epitope may be determined by alanine scanning mutagenesis. In one embodiment, a reduction in binding of a CD3 binding domain to mutated CD3 by 20%, 30%, 50%, 80% or more indicates the amino acid residue of CD3 mutated in an alanine scanning mutagenesis assay is an epitope residue for that CD3 binding domain. Alternatively, a CD3 epitope may be determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography (e.g., crystallography methods).

In some embodiments, the CD3 epitope as determined by crystallography is determined using amino acids Q1-M7 of CD3. In some embodiments, the CD3 epitope as determined by crystallography is determined using amino acids QDGNEEMGGITQTPYK (SEQ ID NO: 107) of CD3.

In some embodiments, the CD3 epitope as determined by crystallography may be performed by combining the anti-CD3 antibody Fab, dissolved in 0.15 M NaCl, 25 mM tris, pH 7.5 at 10 mg/ml, with a 2-fold molar excess (1 mg) of CD3ε peptide and initially screening a sparse matrix of precipitants in a sitting drop vapor diffusion format. Optimized crystals may be grown from a 1:1 mixture with reservoir solution containing 70% v/v methyl-pentanediol, and 0.1 M HEPES buffer at pH 7.5. The reservoir may be be used as a cryoprotectant. The crystals may be transferred to cryogenic temperature by sudden immersion into liquid nitrogen.

The diffraction data for crystals may be collected at Advanced Photon Source beam line 22ID, using a MAR300 CCD detector. The recorded diffractions may be integrated and scaled using the program HKL2000.

The structure may be phased by molecular replacement (MR) method using program Phaser. For example, the MR search model is a Fab subunit derived from a crystal structure of HGFA/Fab complex (PDB code: 2R0L). The CD3ε peptide is built into the structure based on a Fo-Fc map. The structure may be subsequently refined with programs REFMACS and PHENIX using the maximum likelihood target functions, anisotropic individual B-factor refinement method, and TLS refinement method, to achieve convergence.

In some embodiments of any of the anti-CLL-1 antibodies, the CD3 binding domain comprises the hypervariable regions (HVRs) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:91; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:90; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92.

For example, in some embodiments of any of the anti-CLL1 antibodies, the anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) a CD3 binding domain comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:91; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32.

In some embodiments of any of the anti-CLL-1 antibodies, the CD3 binding domain comprises the hypervariable regions (HVRs) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:73; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some instances, the CD3 binding domain may have a heavy chain variable (VH) domain including an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:82 and/or a light chain variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:83. In some instances, the CD3 binding domain may have a VH domain comprising the amino acid sequence of SEQ ID NO:82 and a VL domain comprising the amino acid sequence of SEQ ID NO:83. In a particular instance, the CD3 binding domain can be 40G5c, or a derivative or clonal relative thereof.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:82 and a light chain variable region comprising the sequence of SEQ ID NO:83.

In some embodiments of any of the anti-CLL-1 antibodies, the CD3 binding domain (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:79; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, the anti-CLL-1 antibody comprises a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32.

In some embodiments of any of the anti-CLL-1 antibodies, the CD3 binding domain (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:79; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81. In some instances, the CD3 binding domain may have a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:84 and/or a VL domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:85. In some instances, the CD3 binding domain may have a VH domain comprising the amino acid sequence of SEQ ID NO:84 and a VL domain comprising the amino acid sequence of SEQ ID NO:85. In a particular instance, the CD3 binding domain can be 38E4v1, or a derivative or clonal relative thereof.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:85.

In some embodiments of any of the anti-CLL-1 antibodies, the CD3 binding domain (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:79; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some instances, the CD3 binding domain may have a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:84 and/or a VL domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:93. In some instances, the CD3 binding domain may have a VH domain comprising the amino acid sequence of SEQ ID NO:84 and a VL domain comprising the amino acid sequence of SEQ ID NO:93. In a particular instance, the CD3 binding domain can be 38E4v11, or a derivative or clonal relative thereof.

For example, provided herein are anti-CLL1 antibodies comprising (i) a CLL-1 binding domain comprises the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (ii) the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-CLL-1 antibody comprises (i) a CLL-1 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32 and (ii) a CD3 binding domain comprising a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:93.

In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is a chimeric anti-murine CD3 binding arm. In some embodiments, the chimeric anti-murine CD3 bind arm is 2C11 (Leo et al. Proc Natl Acad Sci USA. 84: 1374-1378, 1987). In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is a mouse anti-human CD3 binding arm. In some embodiments, the mouse anti-human CD3 binding arm is SP34 (Pessano et al. The EMBO Journal. 4: 337-344, 1985). In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is OKT3. (Fernandes, R. A. et al. 2012. J. Biol. Chem. 287: 13324-13335). In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is UCHT1 (Id.). In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is Muromonab-CD3 (CAS Registry Number: 140608-64-6). In some embodiments, the CD3 binding arm of the anti-CLL1 antibody is described in US Patent Application Publication 2010/0150918.

c) Multispecific Anti-CLL1 Antibodies

In certain embodiments, an anti-CLL-1 antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CLL-1 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CLL-1. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CLL-1. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991). Multispecific antibodies may also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see eg., WO2013026833, WO2009/080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576A1, WO2013026833, and WO2012073985).

The anti-CLL-1 antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to CLL-1 as well as another, different antigen (see, US 2008/0069820, for example).

In a further aspect, the CLL-1 binding domain, CD3 binding domain and/or CLL-1 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-6 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM, and optionally is 10⁻¹³ M. (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]. antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000, BAICORE®-T200 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at ^(˜)10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CMS, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (^(˜)0.2 μM) and/or HBS-P (0.01 M Hepes pH7.4, 0.15M NaCl, 0.005% Surfactant P20) before injection at a flow rate of 5 μl/minute and/or 30 μl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In some aspects the anti-CLL-1 antibody (e.g., anti-CLL-1 TDB antibody) comprises an Fc region comprising an N297G mutation. In some embodiments, the anti-CLL-1 antibody comprises an L234A, L235A, and P329G mutation at amino acid residues 234, 235, and 329 according to EU numbering.

In some embodiments, the anti-CLL-1 antibody comprising the N297G mutation comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH1₁) domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, a second CH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3 (CH3₂) domain. In some instances, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some instances, the CH3₁ and CH3₂ domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH3₁ domain is positionable in the cavity or protuberance, respectively, in the CH3₂ domain. In some instances, the CH3₁ and CH3₂ domains meet at an interface between said protuberance and cavity. In some instances, the CH2₁ and CH2₂ domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH2₁ domain is positionable in the cavity or protuberance, respectively, in the CH2₂ domain. In other instances, the CH2₁ and CH2₂ domains meet at an interface between said protuberance and cavity. In some instances, the anti-CD3 antibody is an IgG1 antibody.

In some embodiments of any of the anti-CLL-1 antibodies, (a) the CD3 binding domain comprises a Fc domain, wherein the Fc domain comprises T366S, L368A, Y407V, and N297G substitution mutations according EU numbering and (b) the CLL-1 binding domain comprises a Fc domain, wherein the Fc domain comprises T366W and N297G substitution mutations according EU numbering. In some embodiments of any of the anti-CLL-1 antibodies, (a) the CD3 binding domain comprises a Fc domain, wherein the Fc domain comprises L234A, L235A, P329G, T366S, L368A, and Y407V, substitution mutations according EU numbering and (b) the CLL-1 binding domain comprises a Fc domain, wherein the Fc domain comprises L234A, L235A, P329G, and T366W substitution mutations according EU numbering.

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-CLL-1 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an anti-CLL-1 antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

C. Assays

Anti-CLL-1 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

Anti-CLL-1 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

In one aspect, an antibody and/or binding domain of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, BIACore®, FACS, or Western blot.

In another aspect, competition assays may be used to identify an antibody and/or binding domain that competes with any of the antibodies described herein for binding to CLL-1 or CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an antibody described herein. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized CLL-1 or CD3 is incubated in a solution comprising a first labeled antibody that binds to CLL-1 or CD3 (e.g., any of the antibodies described herein) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CLL-1 or CD3. The second antibody may be present in a hybridoma supernatant. As a control, immobilized CLL-1 or CD3 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to CLL-1 or CD3, excess unbound antibody is removed, and the amount of label associated with immobilized CLL-1 or CD3 is measured. If the amount of label associated with immobilized CLL-1 or CD3 is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to CLL-1 or CD3. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-CLL-1 antibodies (e.g., anti-CLL-1/CD3 TDB antibody) thereof having biological activity. Biological activity may include, e.g., the ability to inhibit cell growth or proliferation (e.g., “cell killing” activity), the ability to induce cell death, including programmed cell death (apoptosis), or antigen binding activity. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In some embodiments, the activity comprises ability to support target cell (e.g., CLL-1 positive cells) killing and/or the activation of the cytotoxic T cells. In certain embodiments, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) of the invention is tested for such target cell (e.g., CLL-1 positive cells) killing and/or the activation of the cytotoxic effect of T cells biological activity by any of the methods described herein, in particular the Examples. In some embodiments, the target cells such as AML tumor cell lines, PBMC, or AML patient bone marrow cells, may be pre-incubated for 1-2 hours at 37° C. in RPMI medium containing 10% FCS, 2 mM Glutamine and 0.3 mg/ml purified low endotoxin human IgG (Molecular Innovations, HU-GF-ED) to prevent non-specific binding of the TDB to cells with surface expression of FcγRs. In some embodiments, PBMCs may be isolated from normal human donors, and untouched CD8+ T cells in the PBMCs were enriched using the kit from Miltenyi Biotec GmbH (Miltenyi; 130-096-495). The assay may be performed in a 96-well round bottom plate (Costar 3799) containing 60,000 cells/well of human CD8+ T cells and 20,000 cells/well of hIgG-blocked target cells; the effector to target ratio (E:T) was 3:1. The anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) may be added as a 10× working solution serially diluted 3-fold to a final assay concentration spanning 0-10 μg/ml or 0-1 μg/ml. The order of addition may be as follows, 50 μl of 2× target cells, 11 μl of 10×3-fold serially diluted TDB, and 50 ul of 2× CD8+ T cells. The contents may be mixed on a Titer Plate Shaker (Thermo) at setting 6 for 15 sec and incubated for at 37° C. for about 40 hrs. The plate may be mixed once each day. In some embodiments, depletion of hCLL-1 expressing EOL-1, THP-1, HL-60, Nomo-1, ML-2, PL-21, U937 and Molm-13 cells may be monitored using either an anti-CD123-APC (BD Pharmingen; 560087) or anti-CD33-APC reagent (BD Pharmingen; 551378) and propidium iodide by FACS. Activation of CD8+ T cells may be monitored using a combination of anti-CD8-FITC (BD Pharmingen; 555634), anti-CD69-PE (BD Pharmingen; 555531) and anti-CD25 (BD Pharmingen; 555434).

In some embodiments, human or cynomolgus PBMCs may be isolated by Hypaque-Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare), washed at low speed to remove platelets and resuspended in RPMI containing hIgG to block non-specific binding by TDBs to FcγRs. The assay may be performed as described above, except that PBMCs at a concentration of 200,000/well were incubated with 11 μl of 10×3-fold serially diluted anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). Target cell killing (CD14+ monocytes) and activation of effector T cells (CD8+) may be evaluated at ^(˜)40 hours by FACS. Depletion of hCLL-1 expressing CD14+ monocytes may be monitored using an anti-CD14-APC reagent (Human BD Pharmingen; 555399, Cyno Miltenyi: 130-091-243) and propidium iodide.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In one aspect, anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) provided herein are useful for detecting the presence of CLL-1 in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue. In certain embodiments, such tissues include normal and/or cancerous tissues that express CLL-1 at higher levels relative to other tissues

In one embodiment, an anti-CLL-1 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of CLL-1 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-CLL-1 antibody as described herein under conditions permissive for binding of the anti-CLL-1 antibody to CLL-1, and detecting whether a complex is formed between the anti-CLL-1 antibody and CLL-1. Such method may be an in vitro or in vivo method. In one embodiment, an anti-CLL-1 antibody is used to select subjects eligible for therapy with an anti-CLL-1 antibody, e.g. where CLL-1 is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed or detected according to any of the above embodiments include CLL-1-positive cancers, such as CLL-1-positive AML, CLL-1-positive CML, CLL-1-positive MDS, CLL-1-positive chronic myelomonocytic leukemia, CLL-1-positive APL, CLL-1-positive chronic myeloproliferative disorder, CLL-1-positive thrombocytic leukemia, CLL-1-positive pre-B-ALL, CLL-1-positive preT-ALL, CLL-1-positive multiple myeloma, CLL-1-positive mast cell disease, CLL-1-positive mast cell leukemia, CLL-1-positive mast cell sarcoma, CLL-1-positive myeloid sarcomas, CLL-1-positive lymphoid leukemia, and CLL-1-positive undifferentiated leukemia. In some embodiments, a CLL-1-positive cancer is a cancer that receives an anti-CLL-1 immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than “0,” which corresponds to very weak or no staining in >90% of tumor cells, under the conditions described herein in Example B. In another embodiment, a CLL-1-positive cancer expresses CLL-1 at a 1+, 2+ or 3+ level, as defined under the conditions described herein in Example B. In some embodiments, a CLL-1-positive cancer is a cancer that expresses CLL-1 according to a reverse-transcriptase PCR (RT-PCR) assay that detects CLL-1 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

Certain other methods can be used to detect binding of anti-CLL-1 antibodies to CLL-1. Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, labeled anti-CLL-1 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-CLL-1 antibody, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-CLL-1 antibody which binds a different epitope on the CLL-1 polypeptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-CLL-1 antibodies (e.g., anti-CLL-1/CD3 TDB antibody) provided herein may be used in therapeutic methods.

In one aspect, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use as a medicament is provided. In further aspects, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in treating or delaying progression of a cell proliferative disorder (e.g., cancer and/or AML) is provided. In certain embodiments, an anti-CLL-1 antibody (e.g., anti-CLL-1/anti-CD3 bispecific antibody) for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in a method of treating an individual having a cell proliferative disorder comprising administering to the individual an effective amount of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In further embodiments, the invention provides an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in enhancing immune function in an individual having a cell proliferative disorder (e.g., AML). In certain embodiments, the invention provides an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in a method of enhancing immune function in an individual having a cell proliferative disorder (e.g., AML) comprising administering to the individual an effective of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, and/or kill a target cell (e.g., target cell). An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides for the use of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody)) in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of a cell proliferative disorder (e.g., cancer and/or AML). In a further embodiment, the medicament is for use in a method of treating a cell proliferative disorder (e.g., AML) comprising administering to an individual having a cell proliferative disorder (e.g., AML) an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In a further embodiment, the medicament is for activating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expanding (increasing) an effector cell population, and/or killing target cells (e.g., target cells) in the individual. In a further embodiment, the medicament is for use in a method of enhancing immune function in an individual having a cell proliferative disorder (e.g., AML) or an autoimmune disorder comprising administering to the individual an amount effective of the medicament to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, and/or kill a target cell (e.g., target cell). An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating a cell proliferative disorder (e.g., cancer and/or AML). In one embodiment, the method comprises administering to an individual having such a cell proliferative disorder (e.g., AML) an effective amount of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for enhancing immune function in an individual having a cell proliferative disorder (e.g., AML) in an individual having a cell proliferative disorder (e.g., AML). In one embodiment, the method comprises administering to the individual an effective amount of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, and/or kill a target cell (e.g., target cell). In one embodiment, an “individual” is a human.

An anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) of the invention may be used in, for example, in vitro, ex vivo, and in vivo therapeutic methods. In one aspect, the invention provides methods for inhibiting cell growth or proliferation, either in vivo or in vitro, the method comprising exposing a cell to an anti-CLL-1 antibody thereof under conditions permissive for binding to CLL-1. “Inhibiting cell growth or proliferation” means decreasing a cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death. In certain embodiments, the cell is a tumor cell. In further embodiments, the cell is a monocyte, granulocyte, and/or progenitors of the monocyte/granulocyte lineage. In some embodiments, the cell is positive for the presence of FLT3 internal tandem repeats. In some embodiments, cell is positive for the presence of a MLL-AF9 fusion gene (e.g., MLL-AF9 translocation). In some embodiments, the cell is positive for the presence of a chromosome 11q23 translocation. In some embodiments, the cell is positive for positive for the presence of a translocation t(9;11)(p22;q23).

Any of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) provided herein may be used in methods, e.g., therapeutic methods.

In one aspect, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) provided herein is used in a method of inhibiting proliferation of a CLL-1-positive cell (CLL-1-positive cell proliferative disorder, the method comprising exposing the cell to the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) under conditions permissive for binding of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) to CLL-1 on the surface of the cell, thereby inhibiting the proliferation of the cell. In certain embodiments, the method is an in vitro or an in vivo method. In further embodiments, the cell is a monocyte, granulocyte, and/or progenitors of the monocyte/granulocyte lineage. In some embodiments, the cell is positive for the presence of FLT3 internal tandem repeats. In some embodiments, cell is positive for the presence of a MLL-AF9 fusion gene (e.g., MLL-AF9 translocation). In some embodiments, the cell is positive for the presence of a chromosome 11q23 translocation. In some embodiments, the cell is positive for positive for the presence of a translocation t(9;11)(p22;q23).

Inhibition of cell proliferation in vitro may be assayed using the CellTiter-Glo™ Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, Wis.). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may be conducted in 96- or 384-well format, making it amenable to automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and generation of a luminescent signal produced by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in culture. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is expressed as relative light units (RLU).

In another aspect, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use as a medicament is provided. In further aspects, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in a method of treatment is provided. In certain embodiments, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in treating cell proliferative disorder (e.g., CLL-1-positive cancer) is provided. In certain embodiments, the invention provides an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) for use in a method of treating an individual having a cell proliferative disorder (e.g., CLL-1-positive cancer), the method comprising administering to the individual an effective amount of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides for the use of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cell proliferative disorder (e.g., CLL-1-positive cancer). In some embodiments, the cancer is AML. In a further embodiment, the medicament is for use in a method of treating cell proliferative disorder (e.g., CLL-1-positive cancer), the method comprising administering to an individual having CLL-1-positive cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treating CLL-1-positive cancer. In one embodiment, the method comprises administering to an individual having such CLL-1-positive cancer an effective amount of an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

A cell proliferative disorder (e.g., CLL-1-positive cancer) according to any of the above embodiments may be, e.g., CLL-1-positive AML, CLL-1-positive chronic myeloid leukemia (CML), CLL-1-positive myelodysplastic syndrome (MDS), CLL-1-positive chronic myelomonocytic leukemia, CLL-1-positive APL, CLL-1-positive chronic myeloproliferative disorder, CLL-1-positive thrombocytic leukemia, CLL-1-positive pre-B-ALL, CLL-1-positive preT-ALL, CLL-1-positive multiple myeloma, CLL-1-positive mast cell disease, CLL-1-positive mast cell leukemia, CLL-1-positive mast cell sarcoma, CLL-1-positive myeloid sarcomas, CLL-1-positive lymphoid leukemia, and CLL-1-positive undifferentiated leukemia. In some embodiments, a CLL-1-positive cancer is a cancer that receives an anti-CLL-1 immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than “0,” which corresponds to very weak or no staining in >90% of tumor cells, under the conditions described herein in Example B. In another embodiment, a CLL-1-positive cancer expresses CLL-1 at a 1+, 2+ or 3+ level, as defined under the conditions described herein in Example B. In some embodiments, a CLL-1-positive cancer is a cancer that expresses CLL-1 according to a reverse-transcriptase PCR (RT-PCR) assay that detects CLL-1 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

In some embodiments, cell proliferative disorder according to any of the above embodiments may be, e.g., AML, CML, and/or MDS. In some embodiments, CLL-1-positive cell proliferative disorder is a CLL-1-positive AML, CLL-1-positive CML, CLL-1-positive MDS. In some embodiments, the AML is one or more of AML subtype 1, AML subtype 2, AML subtype 3, AML subtype 4, AML subtype 5, AML subtype 6, and AML subtype 7. In some embodiments, the AML is AML subtype 3 (acute promyelocytic leukemia, APML). In some embodiments, the AML is one or more of AML subtype 1, AML subtype 2, AML subtype 4, AML subtype 5, AML subtype 6, and AML subtype 7, and not AML subtype 3.

In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of a mutation in FLT3, nucleophosmin (NPM1), CCAAT/enhancer binding protein alpha (C/EBPα) (CEBPA), and/or c-KIT. In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of FLT3 internal tandem repeats. In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of FLT3 tyrosine kinase domain point mutations. In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of a mutation in isocitrate dehydrogenase 1 and/or 2 (IDH1 and/or IDH2). In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of a mutation in DNA methyltransferase 3A (DNMT3A). In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is NK-AML positive for the presence of (a) a mutation in NPM1 and FLT3, (b) wild-type NPM1 and mutated FLT3, and/or (c) wild-type NPM1 and FLT3.

In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive cytogenetic abnormality such as one or more of t(15;17), t(8;21), inv(16), t(16;16), t(941)(p22;q23), t(6;9)(p23;q34), inv(3)(q21 q26.2), inv(3;3)(q21;q26.2), t(1;22)(p13;q13), t(8;21)(q22;q22), inv(16)(p13;1q22), t(16;16)(p13.1;q22), and/or t(15;17)(q22;q12). In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of a MLL-AF9 fusion gene (e.g., MLL-AF9 translocation). In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) is positive for the presence of a chromosome 11q23 translocation. In some embodiments, the cell proliferative disorder (e.g., CLL-1-positive cancer) is a cell proliferative disorder (e.g., CLL-1-positive cancer and/or AML) positive for the presence of a translocation t(9;11)(p22;q23).

In some embodiments, the cell proliferative disorder ((e.g., CLL-1-positive cancer and/or AML) refractory and/or resistant to an anti-AML antibody drug conjugate. In some embodiments, the anti-AML antibody drug conjugate is an anti-CD33 antibody drug conjugate, an anti-CLL1 antibody drug conjugate, and/or an anti-CD123 antibody drug conjugate. In some embodiments, the anti-CLL-1 antibody drug conjugate is anti-CLL-1 antibody drug conjugate (e.g., anti-CLL-1 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CLL-1 antibody drug conjugate is anti-CD33 antibody drug conjugate (e.g., anti-CD33 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CD123 antibody drug conjugate is anti-CD123 antibody drug conjugate (e.g., anti-CD123 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CLL-1 antibody drug conjugate is described in any one of U.S. Pat. No. 8,088,378 and/or US 2014/0030280, which are hereby incorporated by reference in their entirety.

An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-CLL-1 antibodies (e.g., anti-CLL-1/CD3 TDB antibody) provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-CLL-1 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-CLL-1 antibodies (e.g., anti-CLL-1/CD3 TDB antibody) provided herein and at least one additional therapeutic agent, e.g., as described below.

In some embodiments of any of the methods, used, and/or pharmaceutical formulations described herein, the anti-CLL-1/CD3 TDB antibody may be used as a treatment induction therapy. In some embodiments of any of the methods, used, and/or pharmaceutical formulations described herein, the anti-CLL-1/CD3 TDB antibody may be used as a consolidation therapy. In some embodiments of any of the methods, used, and/or pharmaceutical formulations described herein, the anti-CLL-1/CD3 TDB antibody may be used as a salvage therapy.

Antibodies (e.g., anti-CLL-1/CD3 TDB antibody) described herein can be used either alone or in combination with other agents in a therapy. For instance, an antibody (e.g., anti-CLL-1/CD3 TDB antibody) described herein may be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anthracycline. In some embodiments, the anthracycline is daunorubicin or idarubicin. In some embodiments, the additional therapeutic agent is cytarabine. In some embodiments, the additional therapeutic agent is cladribine. In some embodiments, the additional therapeutic agent is fludarabine or topotecan. In some embodiments, the additional therapeutic agent is a hypomethylating agent such as 5-azacytidine or decitabine or guadecitabine. In some embodiments, the additional therapeutic agent is ATRA (all-trans retinoic acid). In some embodiments, the additional therapeutic agent is arsenic trioxide (also known as trisenox). In some embodiments, the additional therapeutic agent is daunorubicin hydrochloride (also known as cerubidine or rubidomycin). In some embodiments, the additional therapeutic agent is cyclophosphamide (also known as clafen, cytoxan, or neosar). In some embodiments, the additional therapeutic agent is cytarabine (also known as cytosar-u, tarabine pfs, or Ara-C). In some embodiments, the additional therapeutic agent is doxorubicin hydrochloride. In some embodiments, the additional therapeutic agent is idarubicin hydrochloride (also known as idamycin). In some embodiments, the additional therapeutic agent is thioguanine (also known as tabloid). In some embodiments, the additional therapeutic agent is vincristine sulfate (also known as vincasar pfs). In some embodiments, the additional therapeutic agent is sapacitabine. In some embodiments, the additional therapeutic agent is laromustine. In some embodiments, the additional therapeutic agent is tipifarnib. In some embodiments, the additional therapeutic agent is bortezomib (also known as VELCADE). In some embodiments, the additional therapeutic agent is hydroxyurea. In some embodiments, the additional therapeutic agent is etoposide. In some embodiments, the additional therapeutic agent is mitoxantrone. In some embodiments, the additional therapeutic agent is clofarabine. In some embodiments, the additional therapeutic agent is hydroxyurea. In some embodiments, the additional therapeutic agent is FLT3 inhibitor such as quizartinib or midostaurin. In some embodiments, the additional therapeutic agent is an anti-cancer quinolone derivative. In some embodiments, the additional therapeutic agent is vosaroxin. In some embodiments, the additional therapeutic agent is an IDH1 inhibitor or an IDH2 inhibitor. In some embodiments, the additional therapeutic agent is CHK1 inhibitor. In some embodiments, the CHK1 inhibitor is GDC-0575. In some embodiments, the additional therapeutic agent is a Plk inhibitor such as volasertib.

In some embodiments of any of the methods, the additional therapeutic agent is a BCL2 inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax.

In some embodiments of any of the methods, the additional therapeutic agent is an epigenetic modifier. In some embodiments, the epigenetic modifier is a histone deacetylase inhibitor. In some embodiments, the epigenetic modifier is DNA methyltransferases I inhibitor. In some embodiments, the epigenetic modifier is a histone methyltransferases inhibitor. In some embodiments, the epigenetic modifier is a BET inhibitor. In some embodiments, the BET inhibitor selectively targets the first bromodomain (BD1). In some embodiments, the BET inhibitor selectively targets the second bromodomain (BD2). In some embodiments, the BET inhibitor is one or more of GSK1210151A, GSK525762, OTX-01, TEN-010, CPI-203, and CPI-0610.

In some embodiments, an antibody (e.g., anti-CLL-1/CD3 TDB antibody) described herein may be co-administered with chemotherapeutic agents. In some embodiments, the chemotherapeutic agents are cytarabine, daunorubicin hydrochloride, and etoposide. In some embodiments, the chemotherapeutic agents are daunorubicin hydrochloride and cytarabine. In some embodiments, the chemotherapeutic agents are fludara and oforta. In some embodiments, the chemotherapeutic agents are cytarabine and an anthracycline. In some embodiments, the anthracycline is daunorubicin, idarubicin, doxorubicin, or epirubicin. In some embodiments, the chemotherapeutic agents are mitoxantrone, etoposide, and cytarabine. In some embodiments, the chemotherapeutic agents are cytarabine, an anthracycline, and clofarabine.

In some embodiments, an antibody (e.g., anti-CLL-1/CD3 TDB antibody) described herein may be co-administered with chemotherapeutic agents and granulocyte colony stimulating factor. In some embodiments, the chemotherapeutic agents are fludarabine and cytarabine. In some embodiments, the chemotherapeutic agents are fludarabine, cytarabine, and idarubicin.

In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is stem cell transplant. In some embodiments, the additional therapy may be a separate administration of one or more of the therapeutic agents described above.

In some embodiments of any of the methods, the additional therapeutic agent is a glucocorticoid. In some embodiments, the glucocorticoid is selected from the group consisting of dexamethasone, hydrocortisone, cortisone, prednisolone, prednisone, methylprednisone, triamcinolone, paramethasone, betamethasone, fludrocortisone, and pharmaceutically acceptable esters, salts, and complexes thereof. In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the glucocorticoid is a pharmaceutically acceptable ester, salt, or complex of dexamethasone. In some embodiments, the glucocorticoid is prednisone.

In some embodiments, the additional therapy further comprises an anti-AML antibody drug conjugate. In some embodiments, the anti-AML antibody drug conjugate is an anti-CD33 antibody drug conjugate, an anti-CLL1 antibody drug conjugate, and/or an anti-CD123 antibody drug conjugate. In some embodiments, the anti-CD33 antibody drug conjugate is gemtuzumab ozogamicin (also known as MYLOTARG). In some embodiments, the anti-CLL-1 antibody drug conjugate is anti-CLL-1 antibody drug conjugate (e.g., anti-CLL-1 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CLL-1 antibody drug conjugate is anti-CD33 antibody drug conjugate (e.g., anti-CD33 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CD33 antibody drug conjugate is vadastuximab talirine (also known as SGN-CD33A). In some embodiments, the anti-CD123 antibody drug conjugate is anti-CD123 antibody drug conjugate (e.g., anti-CD123 pyrrolobenzodiazepine (PBD) antibody drug conjugate). In some embodiments, the anti-CLL-1 antibody drug conjugate is described in any one of U.S. Pat. No. 8,088,378 and/or US 2014/0030280, which are hereby incorporated by reference in their entirety.

In some embodiments of any of the methods, the additional therapy comprises cancer immunotherapies. In some embodiments of any of the methods, the cancer immunotherapy comprises a PD-1 axis binding antagonist. In some embodiments of any of the methods, the cancer immunotherapy comprises a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody binds to human PD-1. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody is MDX-1106 (also known as (BMS-936558/ONO-4538, nivolumab or OPDIVO). In some embodiments, the anti-PD-1 antibody is MK-3475 (also known as SCH 900475, pembrolizumab, lambrolizumab, or KEYTRUDA). In some embodiments, the anti-PD-1 antibody is pidilizumab (also known as CT-011).

In some embodiments of any of the methods, the cancer immunotherapy comprises a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody binds to human PD-L1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. Examples of anti-PD-L1 antibodies that can be used in the methods described herein are described in PCT patent application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, which are incorporated herein by reference in their entirety. Further examples of anti-PD-L1 antibodies that can be used in the methods described herein are described in PCT patent application WO 2007/005874, WO 2011/066389, and US 2013/034559, which are incorporated herein by reference in their entirety.

In some embodiments, an anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

Anti-PD-L1 antibodies described in WO 2010/077634 A1 and U.S. Pat. No. 8,217,149 may be used in the methods described herein. In some embodiments, the anti-PD-L1 antibody is BMS-936559. In some embodiments, the anti-PD-L1 antibody is MED14736. In some embodiments, the anti-PD-L1 antibody is MPDL3280A (CAS Registry Number 1380723-44-3). In certain embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO:94 and a light chain variable region sequence of SEQ ID NO:95. In a still further embodiment, provided is an isolated anti-PD-L1 antibody comprising a heavy chain variable region and/or a light chain variable region sequence, wherein:

-   -   (a) the heavy chain sequence has at least 85%, at least 90%, at         least 91%, at least 92%, at least 93%, at least 94%, at least         95%, at least 96%, at least 97%, at least 98%, at least 99% or         100% sequence identity to the heavy chain sequence:         EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISAD         TSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:94),         and     -   (b) the light chain sequence has at least 85%, at least 90%, at         least 91%, at least 92%, at least 93%, at least 94%, at least         95%, at least 96%, at least 97%, at least 98%, at least 99% or         100% sequence identity to the light chain sequence:         DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTIS         SLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:95).

In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein:

-   -   (a) the HVR-H1 sequence is GFTFSX₁SWIH (SEQ ID NO:96);     -   (b) the HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG (SEQ ID NO:97);         and     -   (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:98);     -   further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S.

In one specific aspect, X₁ is D; X₂ is S and X₃ is T, such that

-   -   (a) the HVR-H1 sequence is GFTFSDSWIH (SEQ ID NO:99);     -   (b) the HVR-H2 sequence is AWISPYGGSTYYADSVKG (SEQ ID NO:100);         and     -   (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:98);

In another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein:

-   -   (a) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A (SEQ ID NO:101);     -   (b) the HVR-L2 sequence is SASX₉LX₁₀S, (SEQ ID NO:102); and     -   (c) the HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T (SEQ ID NO:103);     -   wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is A or F;         X₈ is V or L; X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S;         X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V,         P, T or I; X₁₅ is A, W, R, P or T. In a still further aspect, X₄         is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁         is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A, such that     -   (a) the HVR-L1 sequence is RASQDVSTAVA (SEQ ID NO:104);     -   (b) the HVR-L2 sequence is SASFLYS, (SEQ ID NO:105); and     -   (c) the HVR-L3 sequence is QQYLYHPAT (SEQ ID NO:106).

Thus, in certain embodiments, an anti-PD-L1 antibody comprises a heavy chain variable region comprising the following HVR-H1, HVR-H2 and HVR-H3 sequences, and comprises a light chain variable region comprising the following HVR-L1, HVR-L2 and HVR-L3 sequences:

-   -   (a) the HVR-H1 sequence is GFTFSDSWIH (SEQ ID NO:99);     -   (b) the HVR-H2 sequence is AWISPYGGSTYYADSVKG (SEQ ID NO:100);     -   (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:98);     -   (d) the HVR-L1 sequence is RASQDVSTAVA (SEQ ID NO:104);     -   (e) the HVR-L2 sequence is SASFLYS, (SEQ ID NO:105); and     -   (f) the HVR-L3 sequence is QQYLYHPAT (SEQ ID NO:106).

In some embodiments of any of the methods, the cancer immunotherapy therapy comprises a PD-L2 binding antagonist. In some embodiments, the cancer immunotherapy comprises a PD-L2/IgG1 fusion protein (AMP-224).

In some embodiments, the cancer immunotherapy comprises an agonist directed against an activating co-stimulatory molecule. In some embodiments, an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist directed against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the cancer immunotherapy comprises an antagonist directed against an inhibitory co-stimulatory molecule. In some embodiments, an inhibitory co-stimulatory molecule may include CTLA-4 (also known as CD152), TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some embodiments of any of the methods, the cancer immunotherapy comprises CTLA-4 (also known as CD152) inhibition. In some embodiments, the cancer immunotherapy comprises a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010, MDX-101, or Yervoy®). In some embodiments, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206).

In some embodiments, the cancer immunotherapy comprises an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody. In some embodiments, the cancer immunotherapy comprises MGA271 (also known as enoblituzumab).

In some embodiments, the cancer immunotherapy comprises an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299. In some embodiments of any of the methods, the cancer immunotherapy comprises immune agonists.

In some embodiments, the additional therapy comprises adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR) (e.g, CAR T cell). In some embodiments, the additional therapy comprises a CAR T cell directed against WT1. In some embodiments, the CART cell directed against WT1 is WT128z. In some embodiments, the additional therapy comprises a CAR T cell directed against LeY. In some embodiments, the additional therapy comprises a CAR T cell directed against CD33. In some embodiments, the additional therapy comprises a CAR T cell directed against CD123. In some embodiments, the additional treatment comprises adoptive transfer of a T cell comprising a dominant-negative TGF beta receptor, e.g, a dominant-negative TGF beta type II receptor.

In some embodiments, the additional therapy comprises an antagonist directed against CD47. In some embodiments, the antagonist directed against CD47 is an anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is Hu5F9-G4.

In some embodiments, the additional therapy comprises an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In some embodiments, the additional therapy comprises urelumab (also known as BMS-663513). In some embodiments, the additional therapy comprises an agonist directed against CD40, e.g., an activating antibody. In some embodiments, the additional therapy comprises CP-870893 or R07009789. In some embodiments, the additional therapy comprises an agonist directed against OX40 (also known as CD134), e.g., an activating antibody. In some embodiments the additional therapy comprises an agonist directed against CD27, e.g., an activating antibody. In some embodiments, the additional therapy comprises CDX-1127 (also known as varlilumab). In some embodiments, the additional therapy comprises an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is GDC-0919 (also known as NLG919 and RG6078). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In some embodiments, the IDO antagonist is an IDO antagonist shown in WO2010/005958 (the contents of which are expressly incorporated by record herein). In some embodiments the IDO antagonist is 4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (e.g., as described in Example 23 of WO2010/005958). In some embodiments the IDO antagonist is

In some embodiments, the IDO antagonist is INCB24360. In some embodiments, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan).

In some embodiments, the additional therapy comprises an antineoplastic agent. In some embodiments, the additional therapy comprises an agent targeting CSF-1R (also known as M-CSFR or CD115). In some embodiments, the additional therapy comprises an anti-CSF-1R antibody (also known as IMC-CS4 or LY3022855) In some embodiments, the additional therapy comprises an anti-CSF-1R antibody, RG7155 (also known as R05509554 or emactuzumab). In some embodiments the additional therapy comprises an interferon, for example interferon alpha or interferon gamma. In some embodiments, the additional therapy comprises Roferon-A (also known as recombinant Interferon alpha-2a). In some embodiments, the additional therapy comprises GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®). In some embodiments, the additional therapy comprises IL-2 (also known as aldesleukin or Proleukin®). In some embodiments, the additional therapy comprises IL-12. In some embodiments, the additional therapy comprises IL27. In some embodiments, the additional therapy comprises IL-15. In some embodiments, the additional therapy comprises ALT-803. In some embodiments the additional therapy comprises an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518. In some embodiments, the antibody targeting GITR is MK04166 (Merck).

In some embodiments, the additional therapy comprises an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, the additional therapy comprises ibrutinib. In some embodiments, the additional therapy comprises an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapy comprises AG-120 (Agios).

In some embodiments, the additional therapy comprises a cancer vaccine. In some embodiments, the cancer vaccine is a personalized cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine. In some embodiments the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the cancer vaccine is a DNA-based cancer vaccine. In some embodiments, the DNA-based cancer vaccine comprises naked DNA. In some embodiments, the DNA-based cancer vaccine comprises cells transfected with DNA. In some embodiments, the cells are dendritic cells. In some embodiments, the cancer vaccine is an RNA-based cancer vaccine. In some embodiments, the RNA-based cancer vaccine is an mRNA-based cancer vaccine. In some embodiments, the mRNA-based cancer vaccine comprises naked RNA. In some embodiments, the RNA-based cancer vaccine comprises cells transfected with RNA. In some embodiments, the RNA is coated on particles. In some embodiments, the RNA is transfected in dendritic cells in vitro. In some embodiments, the mRNA is tailored to act as an adjuvant to stimulate the innate immune system. In some embodiments, the cancer vaccine comprises an adjuvant. In some embodiments, the adjuvant comprises a TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN. In some embodiments, the cancer vaccine comprises a liposome.

In some embodiments, the additional therapy comprises an IL-10 antagonist. In some embodiments, the additional therapy comprises an IL-4 antagonist. In some embodiments, the additional therapy comprises an IL-13 antagonist. In some embodiments, the additional therapy comprises an IL-17 antagonist. In some embodiments, the additional therapy comprises an HVEM antagonist. In some embodiments, the additional therapy comprises an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS. In some embodiments, the additional therapy comprises a treatment targeting CX3CL1. In some embodiments, the additional therapy comprises a treatment targeting CXCL9. In some embodiments, the additional therapy comprises a treatment targeting CXCL10. In some embodiments, the additional therapy comprises a treatment targeting CCL5. In some embodiments, the additional therapy comprises an LFA-1 or ICAM1 agonist. In some embodiments, the additional therapy comprises a Selectin agonist.

In some embodiments, the additional therapy comprises an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K2). In some embodiments, the additional therapy comprises cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, the additional therapy comprises trametinib (also known as Mekinist®). In some embodiments, the additional therapy comprises binimetinib.

In some embodiments, the additional therapy comprises a delta-selective inhibitor of a phosphatidylinositol 3-kinase (PI3K). In some embodiments, the additional therapy comprises idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the additional therapy comprises taselisib (also known as GDC-0032). In some embodiments, the additional therapy comprises BYL-719.

In some embodiments, the additional therapy comprises an inhibitor of LSD1 (also known as KDM1A). In some embodiments, the additional therapy comprises ORY-1001. In some embodiments, the additional therapy comprises GSK2879552.

In some embodiments, the additional therapy comprises an inhibitor of MDM2. In some embodiments, the additional therapy comprises RG7112. In some embodiments, the additional therapy comprises idasanutlin (also known as RG7388 or R05503781). In some embodiments, the additional therapy comprises DS-3032b. In some embodiments, the additional therapy comprises SAR405838. In some embodiments, the additional therapy comprises CGM-097. In some embodiments, the additional therapy comprises MK-8242. In some embodiments, the additional therapy comprises AMG-232.

In some embodiments, the additional therapy comprises an inhibitor of BCL2. In some embodiments, the additional therapy comprises venetoclax.

In some embodiments, the additional therapy comprises an inhibitor of CHK1. In some embodiments, the additional therapy comprises GDC-0575 (also known as ARRY-575). In some embodiments, the additional therapy comprises GDC-0425 (also known as RG7602). In some embodiments, the additional therapy comprises LY2606368.

In some embodiments, the additional therapy comprises an inhibitor of activated hedgehog signaling pathway. In some embodiments, the additional therapy comprises ERIVEDGE.

In some embodiments, the additional therapy comprises an agent that recruits T cells to the tumor. In some embodiments, the additional therapy comprises Iirilumab (IPH2102/BMS-986015). In some embodiments, the additional therapy comprises Idelalisib.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of a CLL-1/CD3 TDB antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.

In some embodiments of any of the methods, an antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. In some embodiments, the administration is subcutaneous.

Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

As a general proposition, the therapeutically effective amount of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) administered to human will be in the range of about 0.01 to about 100 mg/kg of patient body weight whether by one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example. In one embodiment, an anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody) described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of the anti-CLL-1 antibody (e.g., anti-CLL-1/CD3 TDB antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture may include an anti-CLL-1 antibody wherein the antibody comprises a CLL-1 binding domain of the invention and a CD3 binding domain. In some embodiments, the anti-CLL-1 antibody is a bispecific antibody.

III. Examples

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1-Anti-CLL-1 Antibody

A. Monoclonal Antibody Generation

Monoclonal antibodies against human (hu) and cynomolgus (cyno) CLL-1 were generated using the following procedures by immunizing animals with recombinant hu and cyno CLL-1 extracellular domain (ECD, amino acids of 65-265 huCLL-1 and 65-265 cynoCLL-1) fused to a N-terminal Flag (DYKDDDDK (SEQ ID NO: 108)) expressed in a mammalian expression system. The huCLL1 ECD protein (amino acids 65-265) comprised a SNP, AAA(Lys, K) 244→CAA (GLN, Q), which has a minor allele frequency (MAF) of 29%.

Positive clones were expanded and re-screened for binding to huCLL-1 and cynoCLL-1 by ELISA and FACS. Five clones were identified: m3H10, m6E7, m20B1, m21C9, and m28H12 that reacted strongly by fluorescent activated cell sorting (FACS) with stable cell lines expressing recombinant hu and cyno CLL-1, and with tumor-derived CLL-1 expressed on Acute Myeloid Leukemia tumor cell lines. Alignment of the amino acid sequences of the murine heavy and light variable domains are shown in FIGS. 1A and B. m3H10 and m21C9 share the same heavy and light chain CDRs, only the amino acid sequences of m21C9 heavy and light chain variable region is shown in FIG. 1.

B. Species Cross-Reactivity and Binding Affinity

Monoclonal antibodies were tested to determine if they cross-react with cynoCLL-1 extra-cellular domain (ECD) (which is 85.07% identical and 87.35% similar to the huCLL-1 protein ECD). The chimeric anti-CLL-1 human IgG were captured by mouse anti-human IgG coated on the CM5 sensor chip. For kinetics measurements, three-fold serial dilutions of human or cyno CLL-1 (4.1 nM to 1000 nM) were injected in HBS-EP buffer. Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the ratio koff/kon. Table 2 below shows that the chimeric version of the five antibodies (m3H10, m6E7, m20B1, m21C9, and m28H12) recognized both recombinant hu and cynoCLL-1 and provides details regarding the kinetics of the interaction with hu and cyno-CLL-1. Further confirmation of cross-reactivity to cyno CLL-1 was done by FACS analyses of blood from cynomolgus (Mauritian origin) (data not shown).

TABLE 2 Biacore of Anti-CLL-1 Antibodies Ligand Analyte ka (M⁻¹s⁻¹) kd (s⁻¹) kD (M) ch3H10 huCLL-1-Flag 2.7 × 10⁵ 2.4 × 10⁻³ 8.7 nM CynoCLL-1 Flag 1.7 × 10⁵ 7.7 × 10⁻⁴ 4.3 nM ch6E7 huCLL-1-Flag 4.6 × 10⁵ 4.4 × 10⁻⁴ 0.9 nM CynoCLL-1 Flag 4.0 × 10⁵ 4.6 × 10⁻⁴ 1.1 nM ch20B1 huCLL-1-Flag 2.2 × 10⁵ 1.0 × 10⁻³ 4.5 nM CynoCLL-1 Flag 1.9 × 10⁵ 1.2 × 10⁻³ 6.1 nM ch21C9 huCLL-1-Flag 2.5 × 10⁵ 2.4 × 10⁻³ 9.7 nM CynoCLL-1 Flag 1.6 × 10⁵ 1.2 × 10⁻³ 7.1 nM ch28H12 huCLL-1-Flag 5.0 × 10⁵ 9.5 × 10⁻³  18 nM CynoCLL-1 Flag 6.7 × 10⁵ 2.3 × 10⁻⁴ 0.3 nM

Scatchard analysis was performed following standard procedures (Holmes et al., Science 256:1205-1210 (1992)) to determine the relative binding affinities of the antibodies including ch6E7 and ch21C9.

Anti-CLL-1 antibodies were [I¹²⁵] labeled using the indirect lodogen method. The [I¹²⁵] labeled anti-CLL-1 antibodies were purified from free ¹²⁵I-Na by gel filtration using a NAP-5 column (GE Healthcare); the purified iodinated anti-CLL-1 antibodies had a range of specific activities of 8-10 μCi/μg. Competition assay mixtures of 50 μL volume containing a fixed concentration of [I¹²⁵] labeled antibody and decreasing concentrations of serially diluted, unlabeled antibody were placed into 96-well plates. HEK293AD cells stably expressing recombinant hu or cynoCLL-1 or HL-60 tumor cells expressing endogenous CLL-1 were cultured in growth media at 37° C. in 5% CO₂. Cells were detached from the flask using Sigma Cell Dissociation Solution and were washed with binding buffer, which consisted of Dulbecco's Modified Eagle Medium (DMEM) with 1% bovine serum albumin (BSA), 300 mM human IgG and 0.1% sodium azide. The washed cells were added to the 96 well plates at a density of 100,000 cells in 0.2 mL of binding buffer. The final concentration of the [I¹²⁵] labeled antibody in each well was ^(˜)250 pM. The final concentration of the unlabeled antibody in the competition assay ranged from 1000 nM through ten 2-fold dilution steps to a 0 nM buffer-only assay. Competition assays were carried out in triplicate. Competition assays were incubated for 2 hours at room temperature. After the 2-hour incubation, the competition assays were transferred to a Millipore Multiscreen filter plate (Billerica, Mass.) and washed 4 times with binding buffer to separate the free from bound [I¹²⁵] labeled antibody. The filters were counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.). The binding data was evaluated using NewLigand software (Genentech), which uses the fitting algorithm of Munson and Robard to determine the binding affinity of the antibody (Munson and Robard 1980).

Table 3 shows the affinity (kD range of 0.45-1.2 nM) to recombinant hu and cynoCLL-1 expressed by HEK293AD CLL-1 stable cells and to HL-60 cells.

TABLE 3 Antibody Affinity [kD = nM] to CLL-1 (Scatchard Analysis). Cells ch6E7 ch21C9 HL-60 K_(D) (nM) 0.65 0.45 EOL-1 K_(D) (nM) 293AD/huCLL-1 K_(D) (nM) 0.80 0.59 293AD/cynoCLL-1 K_(D) (nM) 1.0 1.2

C. Monoclonal Antibody Epitope Grouping

Epitope grouping was also determined using a cell-based competition binding FACS assay. HL-60 cells were pre-incubated with or without 50-100 fold excess of unlabeled competing antibodies, then stained with directly labeled detection antibodies, a reduction of the signal from detecting antibody indicating that the unlabeled competing antibody binds to the same or similar region on CLL-1 as the detecting antibody—this should occur when the same antibody is used as both detector and competitor. When there is no blocking of detector signal by a different unlabeled antibody, the unlabeled antibody is binding to a different region in CLL-1.

TABLE 4 Anti-CLL-1 Competition Experiments Competing antibodies Detecting antibodies ch6E7 ch20B1 ch21C9 ch28H12 R&D R&D Systems-PE ✓ X ✓ X ✓ (Clone 687317) ch6E7-DyLight650 ✓ X n/a X n/a ch28H12-DyLight650 n/a X n/a ✓ n/a ch21C9-DyLight650 ✓ X ✓ X ✓ eBioscience HB3-PE X X X X ✓ BD Biosciences 50C1-PE X X X X X

Table 4 shows epitope grouping of the antibodies to CLL-1. ch6E7 and ch21C9, but not ch20B1 and ch28H12, bin with R&D Systems-PE (Clone 687317). R&D Systems also blocked eBioscience clone HB3, but ch6E7 and ch21C9 were unable to block eBioscience clone HB3 binding. ch20B1 and ch28H12 failed to compete with any other antibody suggesting each antibody binds a distinct epitope. All antibodies failed to compete with BD Biosciences clone 50C1 also suggesting that it binds a distinct epitope.

D. Humanization of anti-CLL-1 Antibodies

Monoclonal antibody 6E7 and 21C9 was humanized as described below. Residue numbers are according to Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

Variants constructed during the humanization of 6E7 and 21C9 were assessed in the form of an IgG. The VL and VH domains from murine 6E7 and 21C9 were aligned with the human VL kappa I (VLKI) and human VH subgroup I (VHI) consensus sequences. Hypervariable regions from the murine antibodies were engineered into VLKI and VHI acceptor frameworks. Specifically, from the mu6E7 and mu21C9 VL domain, positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) were grafted into VLKI and from the mu6E7 and mu21C9 VH domain, positions 26-35 (H1), 50-65 (H2) and 93-102 (H3) were grafted into VHI.

The binding affinity of the antibodies in this section was determined by BIAcore™ T200 Format. Briefly, BIAcore™ research grade CM5 chips were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents according to the supplier's instructions. Goat anti-human Fc IgGs were coupled to the chips to achieve approximately 10,000 response units (RU) in each flow cell. Unreacted coupling groups were blocked with 1M ethanolamine. For kinetics measurements, antibodies were captured to achieve approximately 300 RU. Three-fold serial dilutions of human CLL-1 was injected in HBS-P buffer (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% surfactant P20) at 25° C. with a flow rate of 30 μ/min. Association rates (kon) and dissociation rates (koff) were calculated using a 1:1 Langmuir binding model (BIAcore™ T200 Evaluation Software version 2.0). The equilibrium dissociation constant (Kd) was calculated as the ratio koff/kon.

The binding affinity of the CDR graft humanized 6E7 antibody was compared to chimeric 6E7. Additional variants of the CDR graft humanized 6E7 antibody were made to evaluate the contribution of other vernier positions to binding to CLL-1. For 6E7, initially four additional light chains (L1: CDRs graft+(L4, L48, and K49), L2: CDRs graft+L4, L3: CDRs graft+K49, and L4: CDRs graft+K49) and five additional heavy chains (H1: CDRs graft+(A67, L69, V71, K73), H2: CDRs graft+A67, H3: CDRs graft+L69, H4:CDRs graft+V71, and H5: CDRs graft+K73). K49 on the light chain was the key mouse vernier residue, and L69 and V71 on the heavy chain were determined to be the key mouse vernier residues based on mutational analysis (data not shown). Chimeric 6E7 bound with a KD of 9.59E-10 M, while CDRs graft+K49(LC)+(A67, L69, V71, K73 (HC)), CDRs graft+K49(LC)+(L69, V71 (HC)) bound with a KD of 1.40E-9M, and 1.37E-9M, respectively.

The binding affinity of the CDR graft humanized 21C9 antibody was compared to chimeric 21C9 antibody. Additional variants of the CDR graft humanized 21C9 antibody were made to evaluate the contribution of other vernier positions to binding to CLL-1. For 21C9, initially three additional light chains (L1: CDRs graft+(F36 and S43), L2: CDRs graft+F36, L3: CDRs graft+S43) and five additional heavy chains (H1: CDRs graft+(A67, L69, V71, K73), H2: CDRs graft+A67, H3: CDRs graft+L69, H4:CDRs graft+V71, and H5: CDRs graft+K73). F36 on the light chain was the key mouse vernier residue. Chimeric 21C9 bound with a KD of 8.615E-9 M, while CDRs graft+(F36 and S43(LC))+L69 (HC) and CDRs graft+F36 (LC)+L69 (HC), bound with a KD of 1.053E-8M and 9.785-9M, respectively. L69 on the heavy chain were determined to be the key mouse vernier residues.

The humanized 6E7.L4H1e and 21C9.L2H3 were tested for their ability to bind human and cyno CLL-1 as described above except that cynoCLL-1 replaced huCLL-1 in the cyno binding assay. Binding properties for the humanized antibodies are shown below in Table 5. The binding affinity of the humanized 6E7.L4H1e was 0.34, 0.29, 0.22, and 0.35 Kd (nM) as determined by Scatchard using HL-60, EOL-1, 293AD/cynoCLL1, and 293AD/huCLL-1 cells, respectively. The binding affinity of humanized 21C9.L2H3 was 1.3, 0.74, 2.4, and 3.6 Kd (nM) as determined by Scatchard using HL-60, EOL-1, 293AD/cynoCLL1, and 293AD/huCLL-1 cells, respectively.

TABLE 5 huka cynoka cynokd Antibody huKD (M) (1/Ms) hukd (1/s) cynoKD (M) (1/Ms) (1/s) 6E7.L4H1e 6.218E−10 8.236E+5 5.121E−4 3.170E−10 7.391E+5 2.343E−4 21C9.L2H3 1.171E−8  2.244E+5 2.628E−3 9.472E−9  1.683E+5 1.594E−3

The humanized antibodies 6E7.L4H1e and 21C9.L2H3 were tested under thermal stress (40° C., pH 5.5, 2 weeks) and 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH) Analysis. Samples were thermally stressed to mimic stability over the shelf life of the product. Samples were buffer exchanged into 20 mM His Acetate, 240 mM sucrose, pH 5.5 and diluted to a concentration of 1 mg/mL. One mL of sample was stressed at 40 C for 2 weeks and a second was stored at −70 C as a control. Both samples were then digested using trypsin to create peptides that could be analysed using liquid chromatography (LC)-mass spectrometry (MS) analysis. For each peptide in the sample retention time, from the LC as well as high resolution accurate mass and peptide ion fragmentation information (amino acid sequence information) were acquired in the MS. Extracted ion chromatograms (XIC) were taken for peptides of interest (native and modified peptide ions) from the data sets at a window of +−10 ppm and peaks were integrated to determine area. Relative percentages of modification were calculated for each sample by taking the (area of the modified peptide) divided by (area of the modified peptide plus the area of the native peptide) multiplied by 100.

Both 6E7.L4H1e and h21C9.L2H3 have N⁵⁴G⁵⁵ in DR-H2, which is susceptible to deamination (t0=13.2% and t2wk 14.5% for 6E7.L4H1e and r0=11% and t2wk=11.9%). N54 variants of both antibodies were tested to determine if potential deamination could be reduced without affecting binding to hu and cynoCLL-1. See Table 6.

TABLE 6 huka cynoka cynokd Antibody huKD (M) (1/Ms) hukd (1/s) cynoKD (M) (1/Ms) (1/s) 6E7.L4H1eN54 1.082E−9 9.096E+5 9.837E−4 2.256E−9 8.044E+5 1.815E−3 6E7.L4H1eA54 3.082E−9 7.103E+5 2.189E−3 3.143E−9 6.087E+5 1.913E−3 6E7.L4H1eE54 5.090E−9 4.882E+5 2.485E−3 4.256E−9 6.641E+5 2.827E−3 6E7.L4H1eS54 1.413E−8 5.098E+5 7.205E−3 6.371E−9 5.133E+5 3.270E−3 6E7.L4H1eD54 1.132E−7 3.044E+5 3.444E−2 4.870E−8 1.785E+5 8.694E−3 21C9.L2H3N54 1.510E−8 1.889E+5 2.853E−3 9.302E−9 2.358E+5 2.194E−3 21C9.L2H3S54 2.859E−7 1.416E+5 4.047E−2 5.669E−6 3656 2.072E−2 21C9.L2H3A54 6.215E−7 1.113E+5 6.915E−2 4.818E−5 445.3 2.146E−2 21C9.L2H3E54 8.625E−7 1.022E+5 8.816E−2 4.961E−5 747.5 3.709E−2 21C9.L2H3D54 8.017E−7 2.858E+5 2.291E−2 2.172E−7 4.072E+4 8.846E−3

For the humanized 6E7.L4H1e CDR-H2 N54 antibody variants, A54 had acceptable binding characteristics which were most similar to N54. For the humanized 21C9.L2H3 CDR-H2 N54 antibody variants, all the variants showed a drop in affinity to huCLL-1 in off-rate (10-30 fold) and cynoCLL-1 in on rate (60-500 fold). The binding affinity of the humanized 6E7.L4H1e was 0.67, 0.68, 0.6, and 0.25 Kd (nM) as determined by Scatchard using 293AD/cynoCLL1, 293AD/huCLL-1, HL-60, and EOL-1 cells, respectively. The binding affinity of humanized 6E7.L4H1eN54A was 0.9, 0.89, 0.64, and 0.32 Kd (nM) as determined by Scatchard using 293AD/cynoCLL1, 293AD/huCLL-1, HL-60, and EOL-1 cells, respectively. Alignment of the heavy and light variable domain amino acid sequences of humanized 6E7 and 21C9 antibodies are shown in FIG. 2A-B and FIG. 3A-B, respectively.

E. Epitope Mapping

To determine the binding epitope of the CLL-1, examination of (a) free antigen CLL-1 and (b) three different antigen-mAb complexes using hydroxyl radical footprinting (HRF) techniques was performed. The samples were exposed to hydroxyl radicals for intervals of 0, 10, 15, and 20 milliseconds (ms) using the X28c Beam line at the Brookhaven National Laboratory. The labeled samples were subjected to deglycosylation using PNGase F. A pilot experiment was first carried out on the deglycosylated samples for optimizing the experimental protocol. The pilot investigation using MS revealed that the samples contained significant amount of polymer contamination, requiring additional clean up. In order to remove the polymer contamination, the samples were precipitated using Trichloroacetic acid in acetone, and subjected to LC-MS analysis. The precipitation step was successful, and the polymer contamination signal in the MS was significantly attenuated. The cleaned samples were subjected to reduction and alkylation, digestion using Trypsin, followed by liquid chromatography coupled with high-resolution mass spectrometry (LC-MS). The MS data was analyzed using ProtMapMS, resulting in dose response plots for each peptide. Results from the free antigen were compared against each of the complex forms. A homology-based model of the antigen was generated using Swiss-Model software, and the solvent protected regions were mapped for each of the three complexes.

The overall sequence coverage obtained using Trypsin mapping was 90.05%. The missing regions were comprised primarily of tryptic peptides that were shorter than 4 residues in length, which can be inherently difficult to detect due to their weak retention properties on the LC column. The HRF process introduces stable side chain oxidative modifications resulting in specific mass shifts, which were identified from the tandem mass spectrometry data. The selected ion chromatograms (SIC) were extracted and integrated for the unoxidized and all oxidized forms of peptide ion (with particular m/z). These peak area values were used to characterize reaction kinetics in the form of dose response (DR) plots, which measure the loss of intact peptide as a function of the hydroxyl radical exposure. The solvent protected regions in the complex experience gradual oxidation reaction as opposed to the free antigen. Differences in the rate of oxidation (called rate constant, RC) serve to highlight the location of the epitope.

ProtMapMS was used to process the MS data, resulting in RC values for each peptide. Final results are shown in Table 6. Peptide location and the corresponding sequence are shown in columns 1 and 2. The third column shows the protection ratio, PR (=RCAntigen/RCComplex) for complex 1 (6E7.L4H1eA54 antibody and CLL-1 antigen). Similarly, fourth and fifth columns show the corresponding protection ratios for complex 2 (21C9.L2H3 antibody engineered with a light chain comprising a cysteine residue at K149 according to Kabat numbering (K149C) and CLL-1 antigen) and complex 3 (R&D Systems monoclonal anti-CLL1 antibody (Clone 687317) and CLL-1 antigen). If the PR value for a given peptide for a particular is less than 1, the corresponding region experienced gain in solvent accessibility due to structural changes introduced during complex formation. A PR value close to 1 indicates that the solvent accessibility of the region remains unchanged, while a PR>1 suggests that the corresponding region exhibits protection from the solvent as a function of the complex formation. The PR values for most of the peptides for each complex are close to 1, indicating minimal change in solvent accessibility for the corresponding regions. Peptide 142-158 consistently shows the highest PR value for all three antibodies, implying significant protection for the region. In addition to protection of the peptide 142-158, the R&D Systems monoclonal anti-CLL1 antibody (Clone 687317, unlike 6E7.L4H1eAG and 21C9.L2H3, also showed significant protection of the region 103-116 as evidenced by the overlapping peptides 103-116 and 105-116.

TABLE 6 Pep Iocn SEQ of SEQ ID ID RCA/R RCA/R RCA/R NO: 1 Sequence NO: C1 C2 C3  65-69 DYKDDDDKLEHVTLK 52 1.4 1.0 1.0  68-69 DDDDKLEHVTLK 53 1.1 0.9 0.8  75-87 MNKLQNISEELQR 54 1.4 1.1 0.90  78-87 LQNISEELQR 55 1.3 1.0 0.8  88-102 NISLQLMSNMNISNK 56 1.1 0.5 0.5 103-116 IRNLSTTLQTIATK 50 1.1 0.8 2.1 105-116 NLSTTLQTIATK 51 1.2 1.0 2.2 105-119* NLSTTLQTIATKLCR 57 NA NA NA 120-124* ELYSK 58 NA NA NA 137-141 WIWHK 59 1.0 0.6 1.3 142-158 DSCYFLSDDVQTWQESK 49 3.1 2.0 3.1 159-160 MACAAQNASLLK 60 1.0 1.2 0.8 171-181 INNKNALEFIK 61 1.7 1.3 1.1 175-181 NALEFIK 62 1.3 1.0 1.3 175-185* NALEFIKSQSR 63 NA NA NA 186-201 SYDYWLGLSPEEDSTR 64 1.0 1.0 1.0 186-204* SYDYWLGLSPEEDSTRGMR 65 NA NA NA 205-117 VDNIINSSAWVIR 66 1.2 1.0 1.0 218-232 NAPDLNNMYCGYINR 67 1.2 1.0 0.9 233-243 LYVQYYHCTYK 68 1.0 1.1 1.0 246-250* MICEK 69 NA NA NA 251-263 MANPVQLGSTYFR 70 0.99 1.1 1.0

F. Internalization of anti-CLL-1 Antibody

To determine whether anti-CLL-1 antibody gets internalized upon binding, HL-60 or HL-60 cells were pre-incubated for 2 hours at 37° C. with 0.3 mg/ml hIgG in RPMI medium to reduce non-specific binding to FcR before seeding in cell culture treated 4-well chamber slides (Nalge Nunc International). Antibody directly conjugated to Dylight 488 at a final concentration of 1 μg/mL was incubated with hIgG-blocked cells on ice for 30 minutes in the dark. The cells were immediately imaged to show membrane staining (T0) and followed with time-lapsed photography over a 10 hour period at 37° C. with a Leica SP5 confocal microscope. A representative example, ch21C9, is rapidly internalized within 30 minutes by HL-60 cells (data not shown). Localization of ch21C9 to the lysosome was confirmed using an in vitro cell-based assay.

Example 2: Efficacy of CLL1 T-Cell Dependent Bispecific (TDB) Antibody

The following examples show that an engineered T-cell dependent bispecific antibody comprising the binding determinants of an anti-human CLL-1 on one arm and an anti-human CD3e on the other arm is a potent immune-modulating molecule that can redirect a persons' immune system to kill Acute Myeloid Leukemia tumor cells and normal CD14+ monocytes from human donors. TDB Efficacy was demonstrated by monitoring the killing of AML tumor cell lines (EOL-1, HL-60, THP-1, U937, Nomo-1, PL-21, ML-2 and Molm-13) by untouched human CD8+ T cells or by monitoring the killing of autologous CD14+ mononuclear cell with autologous T cells in a population of human PBMCs.

The TDB used in the following examples contained the CLL-1 binding determinants from anti-CLL-1 mouse and humanized monoclonal antibody clones from three different epitope bins of 1) 6E7 and/or 21C9, 2) 20B1, and 3) 28H12, and the binding determinants from a humanized anti-CD3ε clone, high affinity antibody 38E4v1 and low affinity antibody 40G5c as determined by Biacore (hCD3ε 1-27 Fc (1.0 and 13 nM respectively), hCD3εγ (0.5 and 12 nM, respectively) and cynoCD3εγ (0.7 and 14 nM, respectively) as well as very high affinity antibody 38E4v11 with a hCD3 (0.05 nM; Fab Biacore measurements to hCD3ε-5.00E+07 ka (1/Ms), 1.60E-03 kd (1/s), and 0.032 KD (nM)). Fab Biarcore measurements to hCD3ε for 38E4v1 were 8.15E+06 ka (1/Ms), 3.17E-03 kd (1/s), and 0.389 KD (nM). 38E4v1, 38E4v11, and 40G5c bind a human CD3ε polypeptide (a fragment of the human CD3ε polypeptide consisting of amino acids 1-26 (SEQ ID NO:86) or 1-27 (SEQ ID NO:87)) and amino acid residue Glu5 of CD3ε is not required for binding (data not shown; see also PCT/US14/70951, which is incorporated by reference in its entirety).

A. Materials and Methods

In all experiments, the target cells, AML tumor cell lines, PBMC, or AML patient bone marrow cells, were pre-incubated for 1-2 hours at 37° C. in RPMI medium containing 10% FCS, 2 mM Glutamine and 0.3 mg/ml purified low endotoxin human IgG (Molecular Innovations, HU-GF-ED) to prevent non-specific binding of the TDB to cells with surface expression of FcγRs. In the first example, PBMCs were isolated from normal human donors, and untouched CD8⁺ T cells in the PBMCs were enriched using the kit from Miltenyi Biotec GmbH (Miltenyi; 130-096-495). The assay was performed in a 96-well round bottom plate (Costar 3799) containing 60,000 cells/well of human CD8⁺ T cells and 20,000 cells/well of hIgG-blocked target cells; the effector to target ratio (E:T) was 3:1. TDB (e.g., h6E7 (6E7.L4H1e)-38E4v1, h21C9 (h21C9.L2H3)-38E4v1 or negative control anti-gD-38E4v1) was added as a 10× working solution serially diluted 3-fold to a final assay concentration spanning 0-10 μg/ml or 0-1 μg/ml. The order of addition was as follows, 50 μl of 2× target cells, 11 ul of 10× 3-fold serially diluted TDB, and 50 ul of 2× CD8⁺ T cells. The contents were mixed on a Titer Plate Shaker (Thermo) at setting 6 for 15 sec and incubated for at 37° C. for about 40 hrs. The plate was mixed once each day. Depletion of hCLL-1 expressing EOL-1, THP-1, HL-60, Nomo-1, ML-2, PL-21, U937 and Molm-13 cells were monitored using either an anti-CD123-APC (BD Pharmingen; 560087) or anti-CD33-APC reagent (BD Pharmingen; 551378) and propidium iodide by FACS. Activation of CD8⁺ T cells was monitored using a combination of anti-CD8-FITC (BD Pharmingen; 555634), anti-CD69-PE (BD Pharmingen; 555531) and anti-CD25 (BD Pharmingen; 555434). Fluoresbrite calibration microspheres (Polysciences, Warrington, Pa.) were added to each sample to ensure acquisition of a consistent number of events. Data analyses was done using Flowjo v9.7.5 and EC50's were determined using the program PRISM-4 (GraphPad Software)

For the second example, human or cynomolgus PBMCs were isolated by Hypaque-Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare), washed at low speed to remove platelets and resuspended in RPMI containing hIgG to block non-specific binding by TDBs to FcγRs. The assay was performed as described above, except that PBMCs at a concentration of 200,000/well were incubated with 11 ul of 10× 3-fold serially diluted TDB. The E:T ratio was not determined and was donor dependent. Target cell killing (CD14⁺ monocytes) and activation of effector T cells (CD8⁺) were evaluated at ^(˜)40 hours by FACS. Depletion of hCLL-1 expressing CD14⁺ monocytes was monitored using an anti-CD14-APC reagent (Human BD Pharmingen; 555399, Cyno Miltenyi: 130-091-243) and propidium iodide. Activation of CD8+ T cells was done as outlined in the section above.

Generation of TDBs—

TDB antibodies were produced as full-length antibodies in the knob-into-hole format as human IgG1, as previously described (Atwell et al. J. Mol. Biol. 270: 26-35, 1997). Half antibodies were expressed in either E. coli or Chinese hamster ovary (CHO) cells, purified by Protein A-affinity chromatography, and the proper half antibody pairs were annealed in vitro as described previously (Spiess et al., Nat. Biotechnol. 2013). If TDB antibody production was carried out in CHO cells, the antibody may include an aglycosylation mutation, for example, at residue N297 (e.g., N297G), such that the TDB antibody was an effector-less variant and unable to initiate antibody-dependent cell-mediated cytotoxicity (ADCC).

After annealing, the anti-CLL-1/CD3 TDB antibodies were purified by Hydrophobic Interaction Chromatography (HIC) and characterized by analytical gel filtration, mass spectrometry, and polyacrylamide gel electrophoresis. The purified antibodies ran as a single peak (>99% of the signal) in gel filtration with less than 0.2% aggregates. No homodimers were detected by mass spectrometry.

B. Results

The efficacy of different anti-CLL1 antibody groups were tested in the TDB format. Bin 1 which includes parental h6E7 and h6E7 variants and parental h21C9, Bin 2 which includes 20B1, and Bin 3 which includes 28H12. As shown in FIG. 4A, parental h6E7 (6E7.L4H1eN54) as well as variant 6E7.L4H1eA54 (Bin 1) in combination with 38E4v1 resulted in significant tumor cell (EOL-1) killing, which was both dose dependent and antigen-specific, compared to the non-targeting anti-gD arm in combination with 38E4v1. Furthermore, the product of deamination version of h6E7 (6E7.L4H1eD54), which has significantly reduced affinity for hCLL-1 (113.2 nM), showed negligible cell killing in combination with 38E4v1. Epitope bin 2-anti-CLL-1 antibody, 20B1, while having a similar affinity for human CLL-1 as 6E7.L4H1eA54 had significantly reduced tumor cell (EOL-1) killing in combination with the same anti-CD3 arm (38E4v1). Epitope bin 3-anti-CLL-1 antibody, 28H12, in combination with 38E4v1 showed negligible tumor cell killing. See FIG. 4A. A summary of the results is provided in Table 7 below. Based on these results, human/cyno CLL-1 epitope bin 1 was further explored.

Cell killing EC50 Name huCLL-1 [nM] cyCLL-1 [nM] (ng/ml) h6E7.L4H1N54 1.1 2.2 14 h6E7.L4H1A54 3.1 3.1 34 H6E7.L4H1D54 113.2 48.7 >1000 20B1 4.5 6.1 531 28H12 18 0.3 >1000

Both h6E7 and h21C9 are part of CLL-1 epitope bin 1. Experiments were performed as shown in FIG. 4B, to test the efficacy of h6E7 and h21C9 anti-CLL-1 TDBs killing either AML tumor cell lines (EOL-1/THP-1) or autologous CD14⁺ cells expressing human CLL-1 from a single donor. Both CLL-1 TDBs, h6E7 (6E7.L4H1e)×38E4v1 and h21C9 (h21C9.L2H3)×38E4v1, showed significant killing of target cells in a dose-dependent manner compared to the negative control TDB, anti-gD×38E4v1, which showed no specific killing or CD8⁺ T cell activation at a maximum TDB concentration of 1 μg/ml. Furthermore, the TDB concentration eliciting maximal killing coincided with maximal CD8⁺ T cell activation (FIG. 4C). The EC50s for killing target cells by h6E7 (6E7.L4H1e)×38E4v1 were 9.1 ng/ml (EOL-1), 5.4 ng/ml (THP-1), 2.8 ng/ml (human CD14+ monocytes), and by h21C9 (h21C9.L2H3)×38E4v1 was 14.8 ng/ml (human CD14+ monocytes).

In experiments using other donors (FIG. 5A), h21C9 (h21C9.L2H3)×38E4v1 showed significant killing of EOL-1 (EC50s of 16.3 ng/ml) and very weak activity on HL-60, whereas h6E7 (6E7.L4H1e)×38E4v1 exhibited significant killing of EOL-1 (EC50 of 7.4 ng/ml) and HL-60 (EC50 of 95.6 ng/ml). CLL-1 expression and killing data from AML cell lines representing different subtypes of the disease is shown on a third donor (FIGS. 5B & C), which included a Bis-Fab TDB lacking the hIgG Fc region, h6E7 (6E7.L4H1e)×38E4v1, on EOL-1 cells (dotted line) has similar killing to h6E7 (6E7.L4H1e)×38E4v1 hIgG TDB. Overall, h6E7 (6E7.L4H1e)×38E4v1 showed good in vitro potency, and was also effective even on the low cell surface CLL-1 expressing cell line, Molm-13.

As shown in FIG. 6A, h6E7 (6E7.L4H1e)×38E4v1 and to a lesser extent h21C9 (h21C9.L2H3)×38E4v1 is capable of killing cynomolgus CD14⁺ cells in the presence of autologous cynomolgus PBMCs containing all subtypes of T cells. In addition, pan-T cells isolated from PBMCs in the presence of EOL-1 target cells and 6E7 (6E7.L4H1e)×38E4v1 showed significant killing of target cells with an EC50 of ^(˜)5 ng/ml, which is similar to that observed with human T cells. The TDB concentration eliciting maximal killing coincided with maximal CD8+ T cell activation (FIG. 6B).

A summary of the average EC50s determined from multiple donors is shown in Table 8. As shown in Table 7 using HL-60 cells, EOL-1 cells, and Human Autologous CD14+, the efficacy of h6E7 (6E7.L4H1e) was approximately 5 to 17 times greater than h21C9 (h21C9.L2H3) with the same anti-CD3 arm (38E4v1).

TABLE 8 6E7 21C9 Disease Model (EC50 [ng/ml]) (EC50 [ng/ml]) U937 Histiocytic Lymphoma (Myeloid Lineage) 2.1* ND HL-60 Acute Promyelocytic Leukemia (M3) 104 (n = 2)  1761 PL-21 Acute Myeloid Leukemia (FLT3 ITD) 5.9* ND NOMO-1 Acute Myeloid Leukemia (M5A; MLL-AF9) 12.2*  ND EOL-1 Eosinophilic Leukemia (M4 eos) 4.1 (n = 5) 51.1 THP-1 Acute Monocytic Leukemia (M5) 3.6 (n = 3) ND ML-2 Acute Myeloid Leukemia (M4; MLL-AF9) 2.5* ND Molm-13 Lowest expressor of CLL-1 23 (<25% ND dead) Human Normal Monocytes 2.8 (n = 3) 14.8 Autologous CD14⁺

In another example, potency of h6E7 (6E7.L4H1e)×38E4v1 TDB was tested on a patient sample with a confirmed diagnosis of AML and significant expression of both CD33 and CLL-1 on CD34⁺ cells by flow cytometry (FIG. 7A). The in vitro killing assay was performed as described above using isolated naïve allogeneic human CD8+ T-cells and frozen density-gradient purified bone marrow cells from an AML patient (Stanford Blood Bank, Stanford, Calif.). The experiment was done in the following medium containing the following supplements to support hematopoietic stem cells/progenitors: Iscove's Modified Dulbecco's Medium (IMDM), 10% heat inactivated FCS, 2 mM Glutamine, 1× StemSpan CC100 cytokine cocktail (StemCell Technologies), 20 ng/ml human GM-CSF, 20 ng/ml human G-CSF and 0.3 mg/ml low endotoxin human IgG (Molecular Innovations, Novi, Mich.). The E:T ratio was 3:1 using 150,000 and 50,000 cells, respectively and performed as described above for ^(˜)40 h.

FIG. 7B shows that h6E7×38E4v1 was able to kill AML blasts in a dose-dependent manner compared to the Ig-matched non-targeting TDB, anti-gD×38E4v1. The number of live AML blasts remained unchanged during the 40 h incubation with the anti-gD×38E4v1 TDB suggesting that killing by h6E7 (6E7.L4H1e)×38E4v1 was TDB-specific not due to the growth conditions.

C. In Vivo Efficacy of Anti-CLL-1 TDB

The T cell dependent bispecific (TDB) antibody used in the following examples comprises the human CLL-1 and human CD3e antigen binding determinants. The anti-human CLL-1 arm comprises either the sequence of 6E7 (6E7.L4H1e) or optimized variants h6E7 (N54-A or -S or -E or -Q or -D) humanized Fab and an anti-human CD3e arm sequence comprising either h40G.5c, 38E4v1, or 38E4v11 (corresponding to either low, high, or very high affinity for hCD3ε) humanized Fab.

In vitro characterization of h6E7N54A (also called 6E7.L4H1eA54) was done as described for the in vitro killing assay above. FIG. 8A-D shows that 6E7.L4H1eA54 TDBs carrying either the low affinity (h40G5c) or high (38E4v1) or very high (38E4v11) affinity hCD3e arm are capable of killing AML tumor cell lines with similar potency in a dose-dependent manner. In addition, in vitro assessment of optimized variants of h6E7.L4H1e N54-A or -S or -E or -Q or -D humanized Fab paired with anti-human CD3ε Fab, comprising either 38E4v1 or h40G5c, was done to determine the potency of the hCLL-1 arm. FIG. 8E-G shows that higher affinity of the h6E7 arm to human CLL-1 correlated with improved in vitro potency compared to the N54D variant-aspartic acid (D) is the product of asparagine (N) deamidation, and this modification of N54 leads to lower affinity binding to CLL-1. The rank order of potency for the h6E7 variants was similar, regardless of whether the human CD3ε arm was 38E4v1 or h40G5c. However, as shown in FIG. 8H-I, utilization of the high affinity anti-human CD3e arm (38E4v1) resulted in elevated levels of T cell activation at lower bispecific antibody concentrations compared to that of the low affinity human CD3 arm, h40G5c. Overall, the EC50s were similar for h6E7 variants (N54-A or -S or -E or -Q) in the presence of 38E4v1, and h6E7 variants (N54-A or -E or -Q) in the presence of h40G5c.

Genetically engineered mice co-expressing human CLL-1 and human CD3ε were characterized and used for testing the efficacy of an anti-human CLL-1 TDB in vivo. Human CLL-1 (CLEC12a) BAC transgenic C57BL/6N mice (Charles River, Wilmington, Mass.) and human CD3ε BAC transgenic C57BL/6N mice were generated at Genentech and maintained in accordance with the American Association of Laboratory Animal Care guidelines. Additionally, double BAC transgenic mice co-expressing both human CLL-1 and human CD3ε was accomplished by breeding of the parental hCLL-1 and hCD3ε BAC transgenic animals. Studies to demonstrate the potency of an anti-human CLL-1× anti-CD3ε TDB in vivo were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Genentech.

Double BAC-Tg (2×BAC-Tg) mice were characterized by flow cytometry and shown to express both transgenes—human CLL-1 and human CD3ε. Blood from transgenic mice was treated with ACK lyses buffer to remove red blood cells and pre-incubated with a blocking solution (5% mouse, 5% rat and 0.2 mg/ml human IgG, 0.5% BSA, 2 mM EDTA in PBS) for 20 minutes on ice to reduce non-specific binding to FcγRs. Confirmation of human CD3ε surface expression on 2×BAC-Tg CD8⁺ effector T cells was determined by staining with a rat anti-mouse CD8-FITC (Miltenyi) and a mouse anti-human CD3-PE (BD Biosciences), and confirmation of human CLL-1 expression on 2×BAC-Tg CD11b⁺ monocytes was demonstrated by staining with a rat anti-mouse CD11b-FITC (BD Biosciences) and variant h6E7A (6E7L4H1eA54-Dylight 650 (Genentech). FIG. 9 shows that blood from double BAC-Tg (hCLL-1/hCD3ε) mice express human CD3 on mouse CD8⁺ T cells, and human CLL-1 on mouse CD11b⁺ monocytes but not mouse granulocytes. In addition, FIG. 10 shows that the level of expression of either transgene is similar to that of their corresponding parent.

To demonstrate that the chimeric CD8⁺ T cells (expressing both mouse and human CD3) were capable of being activated by a TDB comprising an anti-human CD3ε arm, blood was stimulated ex vivo with various concentrations of TDBs for 20-40 hours as described in Section A. In this experiment, PBMCs from 2×BAC-Tg mice were isolated by density gradient centrifugation using Histopaque-1083 (Aldrich-Sigma, St Louis, Mo.) followed by a low speed spin to remove platelets. The final concentration range of TDB was 0.3-3000 ng/ml that also included a no TDB control for each test article. The TDBs used were: anti-gD×38E4v1 (isotype matched negative control) or parental h6E7 (6E7.L4H1eN54)×38E4v1 or variant h6E7N54A (6E7.L4H1eA54)×38E4v1 or variant h6E7N54A (6E7.L4H1eA54)×h40G5c. After either ^(˜)20 or ^(˜)40 hours the cells were stained with anti-mouse CD8-FITC and anti-mouse CD69 (eBioscience, San Diego, Calif.) to quantitate T cell effector activation by flow cytometry. FIG. 11 shows that all CLL-1 specific TBDs were capable of achieving maximal activation at 3000 ng/ml.

In vivo efficacy studies to test the potency of TDB antibodies containing various arm combinations of anti-human CLL-1 and anti-human CD3 were done using the 2×BAC-Tg mice. Mice were randomized into groups of 5 and given a single intravenous dose on Day 0 of either 0.1 mg/kg or 0.5 mg/kg. The mice were randomized into groups as follows: Group 1 (Vehicle; Histidine buffer), Group 2 (anti-gD×38E4v1 @ 0.5 mg/kg), Group 3 (anti-gD×h40G5 @ 0.5 mg/kg), Group 4 (h6E7N54A (6E7.L4H1eA54) X 38E4v1 @ 0.1 mg/kg), Group 5 (h6E7N54A (6E7.L4H1eA54)×38E4v1 @ 0.5 mg/kg), Group 6 (h6E7N54A (6E7.L4H1eA54)×h40G5 @ 0.1 mg/kg), Group 7 (h6E7N54A (6E7.L4H1eA54)×h40G5 @ 0.5 mg/kg), and Group 8 (h6E7N54A (6E7.L4H1eA54)×38E4v11 @ 0.5 mg/kg); the anti-gD TDB represents a non-CLL-1 targeting isotype-matched antibody control. Prior to randomization and dosing, all animals were phenotyped by flow cytometry to confirm that the expected expression profile of both transgenes and that the CD8⁺ cells were not activated as judged by the expression of surface CD69. Activation of CD8⁺ cells and counts of CD11b⁺ monocytes were done by flow cytometry on Day-6, 1, 7, and 14. At the time of analyses, blood from an individual naïve 2×BAC-Tg mouse was used for setting the BD FACS Caliber and for establishing a baseline. At termination (day 15), blood from mice was collected for PK analyses.

FIG. 12 shows that after the day of dosing (Day 1), only animals in Groups 4-8 had significantly elevated levels of CD69 expression. Potency was measured by monitoring depletion of mCD11b⁺/hCLL-1⁺ cells using an anti-mouse CD11b-FITC and an anti-human CLL-1-Alexa 647 antibody (50C1; BD Biosciences, San Jose, Calif.) that was shown not to cross-block our therapeutic antibodies h6E7 or h21C9. Potency correlated with antigen-specific activation of CD8⁺ effector cells, as maximum depletion of hCLL-1⁺ cells was achieved the day after dosing by mice treated with (G5) h6E7N54A (6E7.L4H1eA54)×38E4v1 TDB at 0.5 mg/kg and (G8) h6E7N54A (6E7.L4H1eA54)×38E4v11 TDB at 0.5 mg/kg, and significant depletion in mice treated with (G4) h6E7N54A (6E7.L4H1eA54)×38E4v1 @ 0.1 mg/kg or (G7) h6E7N54A (6E7.L4H1eA54)×h40G5c @ 0.5 mg/kg or (G6) h6E7 (6E7.L4H1eN54)×h40G5c at 0.1 mg/kg (FIG. 13). Over the course of the experiment, anti-CLL-1 specific TDBs at 0.5 mg/kg were most effective, regardless of whether the TDB included either a low, high, or very high affinity hCD3ε binding arm. At Day 14, at least ^(˜)25-50% depletion was maintained by mice in Groups 5 and 7. No activation of CD8⁺ T cells or depletion of hCLL-1⁺ cells was observed in the vehicle or non-CLL-1 targeting isotype matched TDB control groups. In a similar experiment, the potency of parental and variants of h6E7 was very similar (data not shown).

D. In Vitro Safety Assessment of Anti-CLL-1 TDB

Human hematopoietic stem cells (HSC; CD34⁺, CD38⁻) express CD33 but appear not to express CLL-1. Therefore, to determine whether a TDB specific for CLL-1 could be an effective and safer therapeutic than a TDB targeting CD33, human bone marrow cells were treated with anti-gD×38E4v1 TDB or h6E7 (6E7.L4H1e)×38E4v1 TDB followed by enumeration of resultant GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colony-forming units derived from HSCs and progenitor cells. By definition, HSCs possess the property of self-renewal and formation of progentiors and differentiated lineage-specific cells whereas progenitors have the capacity to form colonies of differentiated lineage-specific cells but are incapable of self-renewal. Our phenotyping of HSCs (CD34⁺ and CD38⁻) and progenitor/differentiated (CD34⁺ CD38⁺) cells show only CLL-1 expression by the later compared to CD33 which is detected on CD34⁺/CD38⁻ and progenitor/differentiated cells (FIG. 14).

In the following example, a human Colony-Forming Cell (CFU) assay, MethoCult, (StemCell Technologies, Vancouver, BC, Canada) was used to determine the effect of an anti-human CLL-1 TDB treatment on the ability of human bone marrow-derived HSCs to proliferate and differentiate in culture. In this assay, one may predict that immature hematopoietic cells (HSCs) void of CLL-1 expression should be spared depletion by treatment with a CLL-1 TDB whereas the lineage committed progenitors and differentiated cells expressing CLL-1 on the cell surface should be abolished by the treatment.

Fresh human bone marrow and blood from the same donor was acquired from ALLCELLS (Alameda, Calif.), and processed by density-gradient separation. Human CD8+ T cells were isolated from the blood PBMCs and together with bone marrow leukocytes used in the in vitro killing assay as described in Section A. The effector (CD8+ T cells) to target (BM leukocyutes) ratio was 3 (150,000 cells): 1 (50,000 cells). Target cells were resuspended in Iscove's Modified Dulbecco's Medium (IMDM) containing the following supplements to support HSCs: 10% heat inactivated FCS, 2 mM Glutamine, StemSpan CC100 cytokine cocktail (StemCell Technologies), 20 ng/ml human GM-CSF, 20 ng/ml human G-CSF and 0.3 mg/ml low endotoxin human IgG (Molecular Innovations, Novi, Mich.) and pre-incubated for 1 h at 37° C. to block Fc

Rs before the addition of CD8+ T cells and TDBs. The final concentration of TDB in the 100 ul assay volume was either 5 or 50 ng/ml. The test article was h6E7 (6E7.L4H1e)×38E4v1 TDB and the IgG-matched non-targeting negative control was an anti-human gD×38E4v1 TDB. The cells were incubated for 40 h. Depletion of CD14⁺ monocyte by FACS was checked on two replicates, and the remaining 3 replicates were harvested, washed and resuspended in 3 ml of MethoCult. An aliquot of 1.1 ml was dispensed from a 3 ml syringe with a 16 g blunt end needle into a 35 mm dish (StemCell Technologies); two platings per treatment group. The dishes were incubated for 14 days, and monitored from day 7 through day 14.

The killing assay showed specific depletion of CD14⁺ bone marrow monocytes by the h6E7 (6E7.L4H1e)×38E4v1 TDB at both the 5 and 50 ng/ml concentrations compared to the anti-human gD×38E4v1 negative control TDB (FIG. 15A). This confirmed that the anti-CLL-1 TDB was functional. The enumeration of CFU-GM, -M, and -GEMM was done on day 10 and the results are shown in FIG. 15B. An ^(˜)80% drop in CFU-GM, -M and GEMM was observed for cells treated with h6E7 (6E7.L4H1e)×38E4v1 at 5 ng/ml and 50 ng/ml compared to cells treated with anti-human gD×38E4v1 or no treatment. It is assumed that this loss of CFU represents the depletion of hematopoietic myeloid progenitor and mature lineage differentiated cells that express human CLL-1. The remaining CFUs are mostly likely the result of the self-renewal and differentiation capacity of human CLL-1⁽⁻⁾ HSCs. This result provides evidence that a TDB targeting cells expressing human CLL-1 does not drastically affect the capacity of hematopoiesis. A similar experiment using 200-fold more h6E7 (6E7.L4H1e)×38E4v1 TDB gave similar results. In addition, the regeneration of erythrocytes was accessed at day 14. Treatment with h6E7 (6E7.L4H1e)×38E4v1 TDB showed a slight diminution in CFU-E and BFU-E of ^(˜)25% compared to untreated cells and ^(˜)16% to the anti-human gD×38E4v1 TDB (FIG. 15C). The results suggest that erythroid regeneration by HSCs was mostly intact. Lastly, sorting of human bone marrow CD34⁺ cells expressing either CD33 or CLL-1 and plating in the MethoCult assay showed only reconstitution of CFU-GM, CFU-G, or CFU-M for the CD34^(+/)CLL-1⁺ population whereas the CD34⁺/CD33⁺ population produced additional cell lineages (CFU-E and CFU-GEMM) that is indicative of a more primitive hematopoietic cell (data not shown).

E. In Vivo Pharmacokinetics and Pharmacodynamics of Anti-CLL-1 TDB

Combinations of anti-human CLL-1 h6E7N54A (6E7.L4H1eA54) and anti-human CD3ε (h40G5c or 38E4v1 or 38E4v11) bispecific antibodies were studied in vivo to determine their PK and PD in peripheral blood, bone marrow and spleen. Non-specific and target-specific PK were characterized in SCID beige and 2×BAC-Tg mice, respectively. Mice were randomized into groups of 3 and given a single intravenous dose on Day 0 of 0.5 mg/kg. Mice were assigned to the following groups: Group 1 (h6E7N54A (6E7.L4H1eA54)×h40G5c), Group 2 (h6E7N54A (6E7.L4H1eA54)×38E4v1) and Group 3 (h6E7N54A (6E7.L4H1eA54)×38E4v11). The study using the hCLL-1× hCD3e BAC-Tg mice (2×BAC-Tg) was conducted as described above, and blood and tissues were collected at the following time points: Day −7 (pre-dose), and post dose at 15 min, 2 h, 6 h, Day 1, 7, and 14. All time points, except for the pre-dose, were terminal. Bispecific antibody concentrations and cytokine concentrations were measured in blood. T cell activation and reduction of target cell numbers were measured in blood and tissues. Flow analyses were performed on an 8-color BD LSR Fortessa cell analyzer.

FIG. 17A shows that in SCID mice (void of T cells (CD3) and myeloid cells expressing human CLL-1) the bispecific antibodies (Groups 1-3) have similar antibody concentrations over time compared to non-CLL-1 targeting isotype-matched antibody controls (anti-gD×h40G5c and anti-gD×38E4v1). The PK parameters are summarized in the FIG. 17B, and illustrate that in the absence of target the anti-hCLL-1×anti-CD3ε bispecific antibodies demonstrate similar PK and clearance comparable to anti-gD controls. When these bispecific antibodies were examined in mice expressing both targets (human CD3ε and human CLL-1), Cmax values were comparable between the hCLL-1×anti-CD3ε molecules. However, CD3 arm affinity dependent differences in exposure were observed (FIG. 17C). The h6E7N54A×h40G5c had the slowest clearance (CL=10.6 mL/d/Kg) which translated into higher drug exposure over time (AUC=47.1 d.μg/mL) compared to bispecific antibodies comprising 38E4v1 (CL=23.5 mL/d/Kg, AUC=21.3 d.μg/mL) or 38E4v11 (CL=37.4 mL/d/Kg, AUC=18.1 d.μg/mL) (FIG. 17D).

A reduction in mouse cells expressing human CLL-1 was used as a measure of PD in peripheral blood, bone marrow and spleen. FIG. 18 shows that a single 0.5 mg/kg dose of h6E7A (6E7.L4H1eA54) x h40G5c resulted in maximal target reduction around Day 7 in peripheral blood with a concomitant diminution of target cells in bone marrow and spleen. Similar and more profound effects were seen with h6E7N54A (6E7.L4H1eA54)×38E4v1 (FIG. 19) and h6E7N54A (6E7.L4H1eA54)×38E4v11 (FIG. 20). The upregulation of CD69 on mouse CD8+ cells also correlated with the time point associated with maximal target diminution (FIG. 21). The lack of target cells in blood, bone marrow and spleen suggests that the diminution of target cells expressing hCLL-1 was not because of margination to bone marrow or spleen. Similar results were seen in pooled lymph nodes (data not shown).

In another experiment, 2×BAC-Tg mice were treated with vehicle or anti-PD-L1 (6E11) or h6E7N54A (6E7.L4H1eA54)×h40G5c at 0.1 mg/kg or h6E7N54A (6E7.L4H1eA54)×h40G5c 0.5 mg/kg or a combination of h6E7N54A (6E7.L4H1eA54)×h40G5c and anti-PD-L1. Administration of anti-PD-L1 was given at 10 mg/kg IV on Day 0, and followed by twice weekly IP dosing at 5 mg/kg. The results of two independent in vivo studies show that combination treatment with h6E7N54A (6E7.L4H1eA54)×h40G5c at 0.5 mg/kg and anti-PD-L1 at 10 mg/kg resulted in better overall myeloid reduction compared to mice treated with either single agent (FIG. 22A-B). However, in this mouse model only monocytes and a sub-population of granulocytes, eosinophils, express detectable levels of human CLL-1 by flow cytometry. It is possible that the majority of granulocytes in the 2×BAC-Tg model express human CLL-1, but below the limit of detection by flow cytometry. In cynomolgus monkey and humans, there is significant cell surface expression of CLL-1 on monocytes and granulocytes. The improvement in granulocyte reduction in the 2×BAC-Tg model might be explained by that fact that h6E7N54A (6E7.L4H1eA54)×h40G5c may upregulate PD-L1 on myeloid cells. To address this hypothesis, blood from mice treated with either vehicle or h6E7N54A (6E7.L4H1eA54)×h40G5c at 0.1 mg/kg or h6E7N54A (6E7.L4H1eA54)×h40G5c at 0.5 mg/kg were analyzed at Day 7 (study end) for the presence of cell surface PD-L1 on CD11b⁺ myeloid cells. FIG. 22C-D shows that mice treated with single agent, h6E7N54A (6E7.L4H1eA54)×h40G5c, exhibited significant upregulation of PD-L1 expression on granulocytes compared to the negative control group (Vehicle). A similar response was evident on monocytes. Overall, the level of PD-L1 upregulation was associated with the dose of h6E7N54A (6E7.L4H1eA54)×h40G5c. These results demonstrate that h6E7N54A (6E7.L4H1eA54)×h40G5c has the ability to modulate immune checkpoint molecules like PD-L1, and that combining an anti-PD-L1 therapeutic with an anti-CLL-1 directed bispecific antibody like h6E7N54A (6E7.L4H1eA54)×h40G5c or h6E7N54A (6E7.L4H1eA54)×h38E4v1 or h6E7N54A (6E7.L4H1eA54)×h38E4v11 is another therapeutic strategy for AML.

Example 3: Toxicity, Toxicokinetic (TK), and PD Study in Cynomolgus Monkeys

Anti-CLL-1/CD3 TDB antibodies described above (h6E7A (6E7.L4H1eA54)×h40G5c (low affinity anti-human CD3ε arm) and h6E7A (6E7.L4H1eA54)×h38E4v1 (high affinity anti-human CD3ε arm)) were assessed in cynomolgus monkeys to determine toxicity, toxicokinetics (TK), and pharmacodynamics (PD). This study was conducted using purpose bred, naïve, Macaca fascicularis of Mauritius origin. Animals selected for the study were demonstrated to express CLL-1 on circulating CD11b+ myeloid cells (granulocytes and monocytes) by flow cytometry. Four groups of male cynomolgus monkeys were administered vehicle or anti-CLL-1/CD3 TDB antibody via a single intravenous infusion and studied for 8-29 days for target cell depletion and recovery, as summarized in Table 9.

TABLE 9 Study design of single dose toxicity, TK, and PD study in cynomolgus monkeys Scheduled Affinity Dose necropsy (day post Group Test article (CLL-1/CD3) Regimen (mg/kg) N dose) 1 vehicle — Single dose, 0 3 29 2 h6E7A × h40G5c H/L 1 hr. IV 0.5 3 29 3 h6E7A × h40G5c H/L infusion 0.2 3/3 8/22 4 h6E7A × h38E4v1 H/H 0.5 3 29

Serum was collected at multiple time points and stored at −70 C for ELISA to determine the amount of test article in each serum sample. Serum concentration-time profiles were used to estimate PK parameters using WinNonlin software (Pharsight; Mountain View, Calif.). PD effects were determined by standard hematological assessments (differential leukocyte counts) and by measuring the reduction in CD11b⁺ myeloid cells in the peripheral blood and bone marrow by flow cytometry. Bone marrow flow cytometry was conducted as follows: fresh bone marrow aspirates were processed by gradient centrifugation using 90% Hypaque-Ficoll to remove contaminating red blood cells. Subsequently, purified bone marrow cells were stained with antibodies specific to non-human primate CD4 (Pe-Cy7), CD8 (Pacific Blue), C20 (FITC), CD11b (APC) and CLL-1 (PE) in the presence of the viability dye, 7AAD, using the live cell staining conditions described above. Serum cytokine levels were analyzed with a multiplex assay and serum biochemistry was assessed using standard assays.

Four animals were euthanized prior to their scheduled necropsy (all three animals in Group 4 and one in Group 2). All three animals in Group 4 given the anti-CLL-1/CD3 TDB antibody with the high affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h38E4v1) (0.5 mg/kg) displayed fever and moribund condition that correlated with markedly elevated serum cytokine levels (including TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6). These animals were euthanized on Day 1 (N=1) or 2 (N=2) and had microscopic evidence of circulatory collapse consistent with cytokine release syndrome. Due to this toxicity, the anti-CLL-1/CD3 TDB antibody with the high affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h38E4v1) was not evaluated further.

Animals in Group 2 displayed fever between 4-8 days post dose. One of these animals developed poor appetite and malaise requiring early euthanasia on day 6. A specific cause for the morbidity of this animal was not identified and a lower dose of 0.2 mg/kg anti-CLL-1/CD3 TDB antibody with the low affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h40G5c) was investigated in Group 3. This dose was well tolerated in all animals, with no fever or other clinical signs for the duration of the study. A reduced cytokine response was observed in animals administered the anti-CLL-1/CD3 TDB antibody with the low affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h40G5c), with little elevation in most of the proinflammatory cytokines evaluated compared to those animals administered the anti-CLL-1/CD3 TDB antibody with the high affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h38E4v1). All Group 3 animals survived to the scheduled necropsies on Day 8 (N=3) and Day 22 (N=3).

In general, clinical pathology changes for anti-CLL-1/CD3 TDB antibodies with low and high affinity CD3 arms were similar and consistent with target-related marked decreases in myeloid and lymphoid cells, cytokine release, and an associated acute phase inflammatory response. The Group 4 animals and the animal requiring an unscheduled necropsy in Group 2 had a similar magnitude of hematology (PD) effects but generally more pronounced acute phase response compared to monkeys given ≥0.2 mg/kg of the anti-CLL-1/CD3 TDB antibody with the low affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h40G5c) that survived to scheduled necropsies.

Animals receiving the anti-CLL-1/CD3 TDB antibody (6E7.L4H1eA54)×h40G5c) with the low affinity CD3 arm displayed the expected myeloid cell reduction due to pharmacological activity of the bispecific antibody. There were marked reductions of monocytes and neutrophils predominantly on Day 2-4 and 4-8, respectively (FIGS. 23A and B), that correlated with markedly decreased late stage myeloid cells in the bone marrow and neutrophils in the spleen at day 6 (unscheduled) and day 8 necropsies. Flow cytometry assessment of bone marrow from animals euthanized on day 8 confirmed a significant reduction in total CD11b⁺ myeloid cells (FIG. 23C). In addition, all of the CD11b⁺ cells co-expressed cynomolgus CLL-1 (see FIG. 23D), thus demonstrating target specificity, with a concomitant depletion of both blood and the bone marrow myeloid cells. Circulating lymphocytes were reduced beginning on Day 2 (FIG. 24). There was rebound recovery for neutrophils, monocytes, and lymphocytes by Day 12, with markedly increased neutrophils at 0.2 mg/kg on Day 15 that were considered to be a continuation of the rebound response and correlated with increases in G-CSF that occurred by Day 8. All leukocyte counts for individual animals trended toward or approximated baseline by Day 22 or Day 29 and there was complete recovery of microscopic changes in the spleen and bone marrow by Day 22 and Day 29, respectively.

The reduction in clinical signs, morbidity, and diminished cytokine release compared to the high affinity version indicates the improved safety profile of the anti-CLL-1/CD3 TDB antibody with the low affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h40G5c) compared to the anti-CLL-1/CD3 TDB antibody with the high affinity CD3 arm (h6E7A (6E7.L4H1eA54)×h38E4v1).

Drug exposure was confirmed in all animals in the treatment groups. The concentration-time profile and PK parameter summary are shown in FIG. 25. Dose proportional Cmax was observed for the h6E7A×h40G5c TDB antibody ((6E7.L4H1eA54)×h40G5c) between 0.2 and 0.5 mg/kg. Positive anti-therapeutic antibodies (“ATA”) were confirmed in all samples on day 15. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

SEQ ID NAME SEQUENCE NO Human CLL-1 MSEEVTYADL QFQNSSEMEK IPEIGKFGEK APPAPSHVWR   1 (UniProt No. PAALFLTLLC LLLLIGLGVL ASMFHVTLKI EMKKMNKLQN Q5QGZ9; NCBI Ref. ISEELQRNIS LQLMSNMNIS NKIRNLSTTL QTIATKLCRE NP_612210.4) LYSKEQEHKC KPCPRRWIWH KDSCYFLSDD VQTWQESKMA CAAQNASLLK INNKNALEFI KSQSRSYDYW LGLSPEEDST RGMRVDNIIN SSAWVIRNAP DLNNMYCGYI NRLYVQYYHC TYKKRMICEK MANPVQLGST YFREA Human CLL-1 ECD HVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIA   2 (aa 65-265 of SEQ TKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQ ID NO: 1) NASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAW VIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFRE A Human CLL-1 C-type CPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKINNKNALEFIKS   3 lectin-like domain QSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINR (CTLD) LYVQYYHCTYKKRMICEK (aa 133-250 of SEQ ID NO: 1 Cyno CLL-1 MSEEVTYADLKFQNSSETEKIQEIAKEGGKAPPAPSCVWRPAALFLTVLC   4 LLMLIGLGVLGSMFHITLKTAMKKMNKLQNINEELQRNVSLQLMSNMNSS NKIRNLSTTLQTIATRLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDD VRTWQESRMACAAQNASLLKINNKNALEFIKSQSTSYPYWLGLSPEKDYS YGTSVDDIINSSAWVIRNASDLNNMFCGYINRIYVHYDYCIYRKKMICEK MANPVQLGFIHFREA m6E7-HVR L1 RASQSVSTSSYNYMH   5 6E7L4Hle-HVR L1 6E7L4HleA54-HVR L1 m6E7-HVR L2 YASNLES   6 6E7L4Hle-HVR L2 6E7L4HleA54-HVR L2 m6E7-HVR L3 QHSWEIPLT   7 6E7L4Hle-HVR L3 6E7L4HleA54-HVR L3 m6E7-HVR H1 DYYMH   8 6E7L4Hle-HVR H1 6E7L4HleA54-HVR H1 m6E7-HVR H2 RINPYNGAAFYSQNFKD   9 6E7L4Hle-HVR H2 m6E7-HVR H3 ERGADLEGYAMDY  10 6E7L4Hle-HVR H3 6E7L4HleA54-HVR H3 6E7L4HleA54-HVR H2 RINPYAGAAFYSQNFKD  11 m20B1-HVR L1 SASSSISYMY  12 m20B1-HVR L2 DTSKLAS  13 m20B1-HVR L3 HQRSSWT  14 m20B1-HVR H1 SYDIN  15 m20B1-HVR H2 WIYPGDGTTEYNERFKG  16 m20B1-HVR H3 SYDYDYAMDY  17 m21C9-HVR L1 KASQDVSTAVA  18 21C9.L2H-HVR L1 m21C9-HVR L2 SPSYRYT  19 21C9.L2H-HVR L2 m21C9-HVR L3 QQLYSTPYT  20 21C9.L2H-HVR L3 m21C9-HVR H1 DYYLD  21 21C9.L2H-HVR H1 m21C9-HVR H2 RVNPYNGGTIYNQKFKG  22 21C9.L2H-HVR H2 m21C9-HVR H3 DHYRYDPLLDY  23 21C9.L2H-HVR H3 m28H12-HVR L1 RASQSVSSSSYSYMH  24 m28H12-HVR L2 YASNLES  25 m28H12-HVR L3 QHSWEIPYT  26 m28H12-HVR H1 DTYMH  27 m28H12-HVR H2 RIDPANGDTDYDPKFQG  28 m28H12-HVR H3 SGPPYYVMDY  29 m6E7 V_(L) DIVLTQSPSSLIVSLGQRATISCRASQSVSTSSYNYMHWYQQKPGQPPKL  30 LLKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPL TFGAGTKLEIK m6E7 V_(H) QVQLQQSGPELVKPGASVKISCKASGYSFTDYYMHWVKQSHIKSLEWIGR  31 INPYNGAAFYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCAIER GADLEGYAMDYWGQGTSVTVSS 6E7L4Hle V_(L) DIQMTQSPSSLSASVGDRVTITCRASQSVSTSSYNYMHWYQQKPGKPPKL  32 LIKYASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSWEIPL TFGQGTKVEIK 6E7L4Hle V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQAPGQGLEWIGR  33 INPYNGAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIER GADLEGYAMDYWGQGTLVTVSS 6E7L4HleA54 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQAPGQGLEWIGR  34 INPYAGAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIER GADLEGYAMDYWGQGTLVTVSS m20B1 V_(L) DIVLTQSPAIMSASPGEKVTMTCSASSSISYMYWYQQKPGTSPKRWIYDT  35 SKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSWTFGGGTK LEIK m20B1 V_(H) EVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWLKQRPGQGLEWIGW  36 TYPGDGTTEYNERFKGKATLTADKSSSTAYLQLSSLTSENSAVYFCARSY DYDYAMDYWGQGTSVTVSS m21C9 V_(L) DIQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWFQQKPGQSPKLLIYS  37 PSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQLYSTPYTFGG GTKLEIK m21C9 V_(H) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYLDWVKQSHGESFEWIGR  38 VNPYNGGTIYNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARDH YRYDPLLDYWGQGTTLTVSS 21C9.L2H3 V_(L) DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWFQQKPGKAPKLLIYS  39 PSYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLYSTPYTFGQ GTKVEIK 21C9.L2H3 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLDWVRQAPGQGLEWIGR  40 VNPYNGGTIYNQKFKGRVTLTRDTSTSTAYLELSSLRSEDTAVYYCARDH YRYDPLLDYWGQGTLVTVSS m28H12 V_(L) DIQMTQSPASLAVSLGQRATISCRASQSVSSSSYSYMHWYQQKPGQPPKL  41 LIKYASNLESGVPARFSGRGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTRLEIK m28H12 V_(H) QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGR  42 IDPANGDTDYDPKFQGKATVTADTSSNTAYLQLSSLTSEDTAVYYCTISG PPYYVMDYWGQGTSVTVSS 6E7L4HleE54-HVR H2 RINPYEGAAFYSQNFKD  43 6E7L4HleS54-HVR H2 RINPYSGAAFYSQNFKD  44 6E7L4HleConsensus- RINPYX₁GAAFYSQNFKD, wherein X₁ is A, E, S, or N  45 HVR H2 6E7L4HleConsensus- EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQAPGQGLEWIGR  46 HVR VH INPYX₁GAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIE RGADLEGYAMDYWGQGTLVTVSS, wherein X₁ is A, E, S or N 6E7L4HleConsensus2- RINPYX₂GAAFYSQNFKD, wherein X₂ is A, E, or S  47 HVR H2 6E7L4HleConsensus2- EVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYMHWVRQAPGQGLEWIGR  48 HVR VH INPYX₂GAAFYSQNFKDRVTLTVDTSTSTAYLELSSLRSEDTAVYYCAIE RGADLEGYAMDYWGQGTLVTVSS, wherein X₂ is A, E, or S 40G5c HVR-H1 NYYIH  71 40G5c HVR-H2 WIYPGDGNTKYNEKFKG  72 40G5c HVR-H3 DSYSNYYFDY  73 40G5c HVR-L1 KSSQSLLNSRTRKNYLA  74 38E4v1 HVR-L1 38E4v11 HVR-L1 CD3 consensus HVR- L1 40G5c HVR-L2 WASTRES  75 40G5c HVR-L3 TQSFILRT  76 38E4v11 HVR-L3 38E4v1 HVR-H1 SYYIH  77 38E4v11 HVR-H1 38E4v1 HVR-H2 WIYPENDNTKYNEKFKD  78 38E4v11 HVR-H2 38E4v1 HVR-H3 DGYSRYYFDY  79 38E4v11 HVR-H3 38E4v1 HVR-L2 WTSTRKS  80 38E4v11 HVR-L2 38E4v1 HVR-L3 KQSFILRT  81 40G5c V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ  82 GLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSL RSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS 40G5c V_(L) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ  83 QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQA EDVAVYYCTQSFILRTFGQGTKVEIK 38E4v1 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQ  84 38E4v11 V_(H) GLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSL RSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS 38E4v1 V_(L) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ  85 QKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQA EDVAVYYCKQSFILRTFGQGTKVEIK 1-26 CD3ϵ QDGNEEMGGITQTPYKVSISGTTVILT  86 1-27 CD3ϵ QDGNEEMGGITQTPYKVSISGTTVILTC  87 CD3 consensus HVR- X₃YYIH, wherein X₃ is N or S  88 H1 CD3 consensus HVR- WIYPX₄X₅X₆NTKYNEKFKX₇, wherein X₄ is G or E, X₅ is  89 H2 D or N, X₆ is D or G, and X₇ is D or G CD3 consensus HVR- DX₈YSX₉YYFDY, wherein X₈ is S or G, and X₉ is N or  90 H3 R CD3 consensus HVR- WX₁₀STRX₁₁S, wherein X₁₀ is A or T, and X₁₁ is E or  91 L2 K CD3 consensus HVR- X₁₂QSFILRT, wherein X₁₂ is K or T,  92 L3 38E4v11 V_(L) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ  93 QKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQA EDVAVYYCTQSFILRTFGQGTKVEIK PD-L1 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE  94 WVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSS PD-L1 V_(L) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL  95 LIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYH PATFGQGTKVEIKR PD-L1 HVR-H1 GFTFSX₁SWIH, wherein X₁ is D or G  96 PD-L1 HVR-H2 AWIX₂PYGGSX₃YYADSVKG, wherein X₂ is S or L;  97 X₃ is T or S PD-L1 HVR-H3 RHWPGGFDY  98 PD-L1 HVR-H1 GFTFSDSWIH  99 PD-L1 HVR-H2 AWISPYGGSTYYADSVKG 100 PD-L1 HVR-L1 RASQX₄X₅X₆TX₇X₈A, wherein X₄ is D or V; X₅ is 101 V or I; X₆ is S or N; X₇ is A or F; X₈ is V or L PD-L1 HVR-L2 SASX₉LX₁₀S, wherein X₉ is F or T; X₁₀ is Y or 102 A PD-L1 HVR-L3 QQX₁₁X₁₂X₁₃X₁₄PX₁₅T, wherein X₁₁ is Y, G, F, or 103 S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V, P, T or I; X₁₅ is A, W, R, P or T PD-L1 HVR-L1 RASQDVSTAVA 104 PD-L1 HVR-L2 SASFLYS 105 PD-L1 HVR-L3 QQYLYHPAT 106 

What is claimed is:
 1. An isolated anti-CLL-1 antibody, wherein the antibody comprises: (a) a CLL-1 binding domain, wherein the CLL-1 binding domain comprises six hypervariable regions (HVRs) as follows: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 6, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; or (ii) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; or (iii) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; or (iv) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29; and (b) a CD3 binding domain, wherein the CD3 binding domain comprises six HVRs as follows: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 71, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 72 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 73; or (ii) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 79; or (iii) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:
 79. 2. The anti-CLL-1 antibody of claim 1, wherein the CLL-1 binding domain comprises the following six hypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:45; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
 3. The anti-CLL-1 antibody of claim 2, wherein the CLL-1 binding domain comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:9.
 4. The anti-CLL-1 antibody of claim 2, wherein the CLL-1 binding domain comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:47.
 5. The anti-CLL-1 antibody of claim 4, wherein the CLL-1 binding domain comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:11.
 6. The anti-CLL-1 antibody of claim 4, wherein the CLL-1 binding domain comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:43.
 7. The anti-CLL-1 antibody of claim 4, wherein the CLL-1 binding domain comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:44.
 8. The anti-CLL-1 antibody of claim 2, wherein the antibody comprises a CLL-1 binding domain comprising: a) a heavy chain variable region comprising the sequence of SEQ ID NO:33 and a light chain variable region comprising the sequence of SEQ ID NO:32; b) a heavy chain variable region comprising the sequence of SEQ ID NO:34 and a light chain variable region comprising the sequence of SEQ ID NO:32; c) a heavy chain variable region comprising the sequence of SEQ ID NO:46 and a light chain variable region comprising the sequence of SEQ ID NO:32; or d) a heavy chain variable region comprising the sequence of SEQ ID NO:48 and a light chain variable region comprising the sequence of SEQ ID NO:32.
 9. The anti-CLL-1 antibody of claim 1, wherein the CLL-1 binding domain comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:20.
 10. The anti-CLL-1 antibody of claim 9, wherein the antibody comprises a CLL-1 binding domain comprising: (a) a heavy chain variable region comprising the sequence of SEQ ID NO:40 and (b) a light chain variable region comprising the sequence of SEQ ID NO:39.
 11. The anti-CLL-1 antibody of claim 1, wherein the antibody comprises a CLL-1 binding domain having one or more of the following characteristics: a. binds to recombinant human CLL-1; b. binds to recombinant cynomolgus monkey CLL-1; c. binds to endogenous CLL-1 on the surface of human peripheral blood mononucleocytes (PBMCs); d. binds to endogenous CLL-1 on the surface of cynomolgus monkey PBMCs; e. binds to endogenous CLL-1 on the surface of a cancer cell; f. binds to endogenous CLL-1 on the surface of an AML cancer cell; g. binds to endogenous CLL-1 on the surface of HL-60 cells; h. binds to endogenous CLL-1 on the surface of EOL-1 cells; i. binds to CLL-1 comprising a K244Q mutation; j. competes for human CLL-1 binding with R&D clone 687317 antibody; k. binds to endogenous human CLL-1 with a Kd of less than 15 nM; l. binds to recombinant human CLL-1 with a Kd of less than 10 nM; and/or m. binds to recombinant cynomolgus monkey CLL-1 with a Kd of less than 10 nM.
 12. The anti-CLL-1 antibody of claim 1, wherein the CD3 binding domain binds a CD3 epitope comprising and/or consisting of Gln1, Asp2, Glu6, and Met7 of human CD3c polypeptide.
 13. The anti-CLL-1 antibody of claim 1, wherein the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76.
 14. The anti-CLL-1 antibody of claim 1, wherein the CD3 binding domain comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:78; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:80; and (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
 76. 15. The anti-CLL-1 antibody of claim 1, wherein the CD3 binding domain comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78 and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 79; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:81.
 16. The anti-CLL-1 antibody of claim 1, wherein the antibody comprises a CD3 binding domain comprising: a) a heavy chain variable region comprising the sequence of SEQ ID NO:82 and a light chain variable region comprising the sequence of SEQ ID NO:83; b) a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:85; or c) a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ ID NO:93.
 17. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody is a monoclonal, humanized, or chimeric antibody.
 18. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody is an IgG antibody.
 19. The anti-CLL-1 antibody of claim 1, wherein the antibody is a bispecific antibody.
 20. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody is an antibody fragment that binds CLL-1 and CD3.
 21. The anti-CLL-1 antibody of claim 20, wherein the anti-CLL-1 antibody fragment is a Fab, Fab′-SH, Fv, scFv, and/or (Fab′)₂ fragment.
 22. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody is a full-length antibody.
 23. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody comprises an aglycosylation site mutation.
 24. The anti-CLL-1 antibody of claim 23, wherein the aglycosylation site mutation is a substitution mutation.
 25. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody comprises reduced effector function.
 26. The anti-CLL-1 antibody of claim 1, wherein the anti-CLL-1 antibody comprises a substitution mutation is at amino acid residue N297, L234, L235, D265, and/or P329G according to EU numbering.
 27. The anti-CLL-1 antibody of claim 26, wherein the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G according to EU numbering.
 28. The anti-CLL-1 antibody of claim 26, wherein the antibody comprises an N297G substitution mutation at amino acid residue 297 according to EU numbering.
 29. The anti-CLL-1 antibody of claim 1, wherein (a) the CD3 binding domain comprises a Fc domain, wherein the Fc domain comprises T366S, L368A, Y407V, and N297G substitution mutations according EU numbering and (b) the CLL-1 binding domain comprises a Fc domain, wherein the Fc domain comprises T366W and N297G substitution mutations according EU numbering.
 30. A pharmaceutical composition comprising the anti-CLL-1 antibody of claim
 1. 31. An isolated anti-CLL-1 antibody, wherein the antibody comprises: (a) a CLL-1 binding domain, wherein the CLL-1 binding domain comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32; or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 32; or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35; or (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 37; or (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40 and a light chain comprising the amino acid sequence of SEQ ID NO: 39; or (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 41; or (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain comprising the amino acid sequence of SEQ ID NO: 32; and (b) a CD3 binding domain, wherein the CD3 binding domain comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 83; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 93; or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:
 85. 